Method for isolating hepatitis C virus peptides

ABSTRACT

Described is a method for isolating Hepatitis C Virus peptides (HPs) which have a binding capacity to a MHC/HLA molecule or a complex comprising said HCV-peptide and said MHC/HLA molecule characterized by the following steps: —providing a pool of HCV-peptide, said pool containing HCV-peptides which bind to said MHC/HLA molecule and HCV-peptides which do not bind to said MHC/HLA molecule, —contacting said MHC/HLA molecule with said pool of HCV-peptides whereby a HCV-peptide which has a binding capacity to said MHC/HLA molecule binds to said MHC/HLA molecule and a complex comprising said HCV-peptide and said MHC/HLA molecule is formed, —detecting and optionally separating said complex from the HCV-peptide which do not bind to said MHC/HLA molecule and optionally isolating and characterizing the HCV-peptide from said complex.

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/EP2003/009482 filed 27 Aug. 2003,which claims priority to Austrian Application No. A 1376/2002 filed 13Sep. 2002, International Application No. PCT/EP03/02005 filed 27 Feb.2003, and European Patent Application No. 03450171.8 filed 11 Jul. 2003,the contents of all of which applications are incorporated herein byreference in their entirety.

The Sequence Listing is submitted on one compact disc (Copy 1), togetherwith a duplicate thereof (Copy 2), each created on Aug. 18, 2005, andeach containing one 344 kb file entitled “SONN059SEQ.txt.” The materialcontained on the compact disc is specifically incorporated herein byreference.

The present invention relates to a method for isolating HCV-peptides,especially for isolating HCV T cell epitopes which have a bindingcapacity to a MHC/HLA molecule.

The immune system is a complex network of inter-related cell types andmolecules, which has evolved in order to protect multicellular organismsfrom infectious microorganisms. It can be divided into the evolutionaryolder innate (or natural) immunity and adaptive (or acquired) immunity.The innate immune system recognizes patterns, which are usually commonand essential for pathogens. For this limited number of molecularstructures germ-line encoded receptors have evolved. By contrast, cellsof the adaptive immune system—B and T lymphocytes—can recognize a hugevariety of antigenic structures. The receptors, termed according to thecell types expressing them, B cell receptor (BCR, its soluble versionsare called antibodies) and T cell receptor (TCR, only cell-surfaceassociated forms) are generated by somatic recombination and show aclonal distribution. Thus, initially there is only small number of cellswith a certain specificity. Upon antigen encounter these cells start todivide (clonal expansion) to generate an effector population able tocope with the antigen. After elimination of antigen a specializedsub-population of cells specifically recognizing this antigen remains asimmunological memory. Taken together the adaptive immune system is slow(compared to innate immunity), however specific and it improves uponrepeated exposure to a given pathogen/antigen.

T cells have a central role in adaptive immunity. Their receptors (TCRs)recognize “major histocompatibility complex” (MHC or HLA):peptidecomplexes on the surface of cells. These peptides are called T cellepitopes and represent degradation products of antigens. There are twomajor classes of T cells: CD8-positive cytotoxic T cells (CTL) arerestricted to MHC class I. CD4-positive helper T cells (HTL) arerestricted to MHC class II. HTL are essential for many features ofadaptive immunity: activation of so called “professionalantigen-presenting cells” (APCs), immunoglobulin (Ig) class switch, thegerminal center reaction and Ig affinity maturation, activation of CTL,immunological memory, regulation of the immune response and others.

MHC molecules collect peptides inside the cell and present them on thecell surface to TCRs of T cells. There are two major classes of MHC,class I recognized by CD8-positive CTL and class II recognized byCD4-positive HTL.

MHC class I molecules consist of a membrane-anchored alpha-chain of 45kDa and the non-covalently attached b2-microglobulin (b2m) of 12 kDA.Resolution of the 3-dimensional structure by X-ray crystallography(Stern and Wiley 1994) revealed that the alpha-chain possesses a cleft,which is closed at both ends and accommodates peptides from 8 to 11amino acids length. Class I molecules are ubiquitously expressed, andthe peptides they present originate from cytoplasmic proteins. These aredegraded by the proteasome, and the resulting peptides are activelytransported into the endoplasmatic reticulum (ER). There, with the helpof several chaperones, MHC:peptide complexes are formed and transportedto the cell surface (Heemels 1995). Thus, MHC class I mirrors theproteome of a cell on its surface and allows T cells to recognizeintracellular pathogens or malignant cells.

MHC class II molecules consist of two membrane-anchored proteins (alpha-and beta-chain) of 35 kDa and 30 kDa, respectively. These together forma cleft, open at both ends, which can accommodate peptides of variablelength, usually from 12 to 25 amino acids. Despite these differences,class I and II molecules share surprising structural similarity (Sternand Wiley 1994). Class II molecules are only expressed on professionalAPC including dendritic cells (DC), B-cells and macrophages/monocytes.These cells are specialized in taking up and processing antigens in theendosomal pathway. Immediately after their biosynthesis, class IImolecules are complexed by the so-called invariant chain (Ii), whichprevents binding of peptides in the ER. When vesicles containing classII:Ii complexes fuse with endosomes containing degradation products ofexogenous antigen, Ii is degraded until the MHC binding cleft is onlycomplexed by the so-called CLIP peptide. The latter is with the help ofchaperones like HLA-DM exchanged by antigenic peptides (Villadangos2000). Finally, MHC:peptide complexes are again presented on the surfaceof APCs, which interact in numerous ways with HTL.

Being both polygenic and extremely polymorphic, the MHC system is highlycomplex. For the class I alpha-chain in humans there are three gene locitermed HLA-A, -B and -C. Likewise, there are three class II alpha-chainloci (DRA, DQA, DPA); for class II beta-chain loci the situation is evenmore complex as there are four different DR beta-chains (DRB1,2,3,5)plus DQB and DPB. Except the monomorphic DR alpha-chain DRA, each genelocus is present in many different alleles (dozens to hundreds) in thepopulation (Klein 1986). Different alleles have largely distinct bindingspecificities for peptides. Alleles are designated, for example,HLA-A*0201 or HLA-DRB1*0401 or HLA-DPA*0101/DPB*0401.

T cell epitopes have been identified by a variety of approaches (Van denEynde 1997). T cell lines and clones have for instance been used toscreen cDNA expression libraries for instance in the context of COScells transfected with the appropriate HLA-molecule. Alternatively,biochemical approaches have been pursued. The latter involved elution ofnatural ligands from MHC molecules on the surface of target cells, theseparation of these peptides by several chromatography steps, analysisof their reactivity with lymphocytes in epitope reconstitution assaysand sequencing by mass spectrometry (Wölfel et al. 1994, Cox et al.1994).

Recently the advent of highly sensitive cytokine detection assays likethe IFN-gamma ELIspot allowed using lymphocytes directly ex vivo forscreening of overlapping synthetic peptides (Maecker 2001, Kern 2000,Tobery 2001). Primarily, Kern et al. (1999&2000) used arrays of pools ofoverlapping 9mer peptides to map CD8+ T cell epitopes in vitro. Later,Tobery et al., 2001 modified this approach and demonstrated that poolscontaining as many as 64 20mer peptides may be used to screen for bothCD8+ and CD4+ T cell epitopes in mice. Both these methods were based onthe monitoring of antigen-specific response by measuring INF-gammaproduction either by intracellular staining (Kern et al 2000) or inELIspot assay (Tobery et al., 2001). By use of mixtures of 15-mers theCD4+ T cell responses are approximately equal to those detected whenwhole soluble protein was used as an antigen, while—not surprising—theCD8+ T cell responses are significantly higher than the often negligibleresponses detected with soluble protein stimulation. Furthermore, theCD8+ T cell responses to a mixture of 15 amino acid peptides are similarto those obtained with a mix of 8-12 amino acid peptides, selected torepresent known MHC class I minimal epitopes. Most probably peptidasesassociated with the cell membrane are responsible for “clipping”peptides to optimal length under these circumstances (Maecker et al,2001).

An interesting alternative is to screen synthetic combinatorial peptidelibraries with specific lymphocytes. For instance, a decapeptide libraryconsisting of 200 mixtures arranged in a positional scanning format, hasbeen successfully used for identification of peptide ligands thatstimulate clonotypic populations of T cells (Wilson, et al., J.Immunol., 1999, 163:6424-6434).

Many T cell epitopes have been identified by so called “Reverseimmunological approaches” Rammensee 1999). In this case the proteingiving rise to a potential T cell epitope is known, and its primarysequence is scanned for HLA binding motifs. Typically dozens to hundredsof candidate peptides or even a full set of overlapping peptides aresynthesized and tested for binding to HLA molecules. Usually, the bestbinders are selected for further characterization with regard to theirreactivity with T cells. This can for instance be done by priming Tcells in vitro or in vivo with the help of HLA transgenic mice.

Hepatitis C Virus (HCV) is a member of the flaviviridiae chronicallyinfecting about 170 million people worldwide. There are at least 6 HCVgenotypes and more than 50 subtypes have been described. In America,Europe and Japan genotypes 1, 2 and 3 are most common. The geographicdistribution of HCV genotypes varies greatly with genotype 1 a beingpredominant in the USA and parts of Western Europe, whereas 1 bpredominates in Southern and Central Europe (Bellentani 2000).

HCV is transmitted through the parenteral or percutan route, andreplicates in hepatocytes. About 15% of patients experience acuteself-limited hepatitis associated with viral clearance and recovery.About 80% of infected persons become chronic carriers. Infection oftenpersists asymptomatically with slow progression for years, howeverultimately HCV is a major cause of cirrhosis, end-stage liver diseaseand liver cancer (Liang 2000). Strength and quality of both HTL and CTLresponses determine whether patients recover (spontaneously or as aconsequence of therapy) or develop chronic infection (Liang 2000).

Standard therapy of HCV comprises a combination of pegylatedinterferon-alpha and the antiviral ribavirin. Virologic responses are,depending on the genotype, achieved in about 50% of HCV patients. Thelow tolerability and the considerable side effects of this therapyclearly necessitate novel therapeutic intervention including therapeuticvaccines (Cornberg 2002). However, presently the detailed understandingof which epitopes in which MHC combination lead to successful immuneresponses is lacking (Ward 2002). Therefore, a comprehensive analysis ofthe T-cell response against the entire HCV is required for developmentof therapeutic epitope-based vaccines.

The HCV virion contains a 9.5-kilobase positive single-strand RNA genomeencoding a large single polyprotein of about 3000 amino acids. Thelatter is processed to at least 10 proteins by both host and HCV-encodedproteolytic activities (Liang 2000). Importantly, the HCV RNA-dependentRNA polymerase is error prone giving rise to the evolution of viralquasispecies and contributing to immune-escape variants (Farci 2000).

It is an object of the present invention to provide a method forscreening HCV-peptides for specific MHC molecules, preferably fordelivering suitable and specific HCV T cell epitopes selected from avariety of HCV-peptides having unknown specificity for a given MHCmolecule and thereby to provide efficient means for preventing andcombatting HCV infections.

Therefore the present invention provides a method for isolatingHCV-peptides which have a binding capacity to a MHC/HLA molecule or acomplex comprising said HCV-peptide and said MHC/HLA molecule whichmethod comprises the following steps:

-   -   providing a pool of HCV-peptides, said pool containing        HCV-peptides which bind to said MHC/HLA molecule and        HCV-peptides which do not bind to said MHC/HLA molecule,    -   contacting said MHC/HLA molecule with said pool of HCV-peptides        whereby a HCV-peptide which has a binding capacity to said        MHC/HLA molecule binds to said MHC/HLA molecule and a complex        comprising said HCV-peptide and said MHC/HLA molecule is formed,    -   detecting and optionally separating said complex from the        HCV-peptides which do not bind to said MHC/HLA molecule and    -   optionally isolating and characterising the HCV-peptide from        said complex.

The present invention also provides a method for isolating HCV T cellepitopes which have a binding capacity to a MHC/HLA molecule or acomplex comprising said epitope and said MHC/HLA molecule which methodcomprises the following steps:

-   -   providing a pool of HCV-peptides, said pool containing        HCV-peptides which bind to a MHC/HLA molecule and HCV-peptides        which do not bind to said MHC/HLA molecule,    -   contacting said MHC/HLA molecule with said pool of HCV-peptides        whereby a HCV-peptide which has a binding capacity to said        MHC/HLA molecule binds to said MHC/HLA molecule and a complex        comprising said HCV-peptide and said MHC/HLA molecule is formed,    -   detecting and optionally separating said complex from the        HCV-peptides which do not bind to said MHC/HLA molecule,    -   optionally isolating and characterising the HCV-peptide from        said complex,    -   assaying said optionally isolated HCV-peptide or said complex in        a T cell assay for T cell activation capacity and    -   providing the optionally isolated HCV-peptide with a T cell        activation capacity as HCV T cell epitope or as complex.

The method according to the present invention enables a screening systemfor screening binding capacity to specific MHC/HLA molecules.Identifying MHC binding molecules is an important tool for molecularcharacterisation of pathogens, tumors, etc. It is therefore possiblewith the present invention to screen a variety (a “pool”) of potentialHCV-peptides as ligands at once for their functional affinity towardsMHC molecules. Binding affinity towards MHC molecules is also anecessary prerequisite for HCV-peptides intended to be used as T cellepitopes, although not a sufficient one. Suitable HCV T cell epitopecandidates have also to be screened and assayed with respect to their Tcell activation capacity. The combination of the screening method forbinding according to the present invention with a suitable T cell assaytherefore provides the method for isolating HCV T cell epitopesaccording to the present invention wherein such T cell epitopes areidentifiable out of a pool of potential HCV-peptides using an MHCbinding assay.

In contrast to the prior art, where such assays have always beenperformed on ligands with known binding/MHC specificity, the methodsaccording to the present invention provide such assays as a screeningtool for pools with ligands of unknown specificity. In the prior artsuch assays have been typically performed on individual single ligands,to test their binding affinity to MHC/HLA molecules. In Kwok et al.(2001) pools of maximally up to 5 overlapping synthetic peptides wereused to generate MHC class II tetramers; the latter were then used tostain PBMC for T cells specific for particular MHC class II:peptidecomplexes which were generated in the binding reaction with the pools of5 peptides. However, an increase in the number of ligands per pool insuch an approach was not regarded as being possible, both forsensitivity and specificity reasons (Novak et al. 2001). A problem withregard to specificity would be the generation of MHC tetramers with morethen one binder per tetramer, if more than one binder would be presentin the pool. This would preclude staining of T cells, which is used foridentification of epitopes in the approach described in the prior art.In strong contrast to that the approach according to the presentinvention allows the identification of more than on binder out of highlycomplex mixtures containing more than one binder.

The nature of the pool to be screened with the present invention is notcritical: the pools may contain any naturally or not naturally occurringHCV-peptide which a) binds specifically to MHC/HLA molecules and/or b)may be specifically recognized by T cells. The binding properties of theset of HCV-peptides of the pool with respect to MHC molecules is notknown; therefore, usually binders and at least a non-binder for a givenMHC molecule are contained in the pool. The pool therefore comprises atleast ten different HCV-peptides. Practically, pools are used accordingto the present invention containing significantly more differentHCV-peptide species, e.g. 20 or more, 100 or more, 1.000 or more or10.000 or more. It is also possible to screen larger libraries (withe.g. more than 10⁶, more than 10⁸ or even more than 10¹⁰ differentHCV-peptide species). This, however, is mainly dependent on theavailability of such HCV-peptide libraries.

Preferred pools of ligands to be used in the method according to thepresent invention are selected from the group consisting of a pool ofpeptides, especially overlapping peptides, a pool of protein fragments,a pool of modified peptides, a pool obtained from antigen-presentingcells, preferably in the form of total lysates or fractions thereof,especially fractions eluted from the surface or the MHC/HLA molecules ofthese cells, a pool comprised of fragments of cells, especially HCVcontaining cells, tumor cells or tissues, especially from liver, a poolcomprised of peptide libraries, pools of (poly)-peptides generated fromrecombinant DNA libraries, especially derived from pathogens or (liver)tumor cells, a pool of proteins and/or protein fragments from HCV ormixtures thereof.

The HCV-peptides of the pools may be derived from natural sources (innative and/or derivatised form) but also be produced synthetically (e.g.by chemical synthesis or by recombinant technology). If (poly)peptideligands are provided in the pools, those peptides are preferablygenerated by peptide synthesizers or by recombinant technology.According to a preferred embodiment, a pool of (poly)peptides may begenerated from recombinant DNA libraries, e.g. derived from HCV or HCVcontaining (tumor) cells, by in vitro translation (e.g. by ribosomedisplay) or by expression through heterologous hosts like E. coli orothers.

The nature of the specific MHC molecules (of course also MHC-likemolecules are encompassed by this term) to be selected for the presentmethods is again not critical. Therefore, these molecules may beselected in principle from any species, especially primates like humans(HLA, see below), chimpanzees, other mammals, e.g. maquaques, rabbits,cats, dogs or rodents like mice, rats, guinea pigs and others,agriculturally important animals like cattle, horses, sheep and fish,although human (or “humanized”) molecules are of course preferred forproviding vaccines for humans. For providing vaccines for specificanimals, especially agriculturally important animals, like cattle,horses, sheep and fish, the use of MHC molecules being specific forthese animals is preferred.

Preferred HLA molecules therefore comprise Class I molecules derivedfrom the HLA-A, -B or -C loci, especially A1, A2, A3, A24, A11, A23,A29, A30, A68; B7, B8, B15, B16, B27, B35, B40, B44, B46, B51, B52, B53;Cw3, Cw4, Cw6, Cw7; Class II molecules derived from the HLA-DP, -DQ or-DR loci, especially DR1, DR2, DR3, DR4, DR7, DR8, DR9, DR11, DR12,DR13, DR51, DR52, DR53; DP2, DP3, DP4; DQ1, DQ3, DQ5, DQ6; andnon-classical MHC/HLA and MHC/HLA-like molecules, which can specificallybind ligands, especially HLA-E, HLA-G, MICA, MICB, Qa1, Qa2, T10, T18,T22, M3 and members of the CD1 family.

According to a preferred embodiment, the methods according to thepresent invention is characterised in that said MHC/HLA molecules areselected from HLA class I molecules, HLA class II molecules, nonclassical MHC/HLA and MHC/HLA-like molecules or mixtures thereof, ormixtures thereof.

Preferably, the optional characterising step of the HCV-peptides of thecomplex is performed by using a method selected from the groupconsisting of mass spectroscopy, polypeptide sequencing, binding assays,especially SDS-stability assays, identification of ligands bydetermination of their retention factors by chromatography, especiallyHPLC, or other spectroscopic techniques, especially violet (UV),infra-red (IR), nuclear magnetic resonance (NMR), circular dichroism(CD) or electron spin resonance (ESR), or combinations thereof.

According to a preferred embodiment the method of the present inventionis characterised in that it is combined with a cytokine secretion assay,preferably with an Elispot assay, an intracellular cytokine staining,FACS or an ELISA (enzyme-linked immunoassays) (see e.g. CurrentProtocols in Immunology).

Preferred T cell assays comprise the mixing and incubation of saidcomplex with isolated T cells and subsequent measuring cytokinesecretion or proliferation of said isolated T cells and/or the measuringup-regulation of activation markers, especially CD69, CD38, ordown-regulation of surface markers, especially CD3, CD8 or TCR and/orthe measuring up-/down-regulation of mRNAs involved in T cellactivation, especially by real-time RT-PCR (see e.g. Current Protocolsin Immunology, Current Protocols in Molecular Biology).

Further preferred T cell assays are selected from T cell assaysmeasuring phosphorylation/de-phosphorylation of components downstream ofthe T cell receptor, especially p56 lck, ITAMS of the TCR and the zetachain, ZAP70, LAT, SLP-76, fyn, and lyn, T cell assays measuringintracellular Ca⁺⁺ concentration or activation of Ca⁺⁺-dependentproteins, T cell assays measuring formation of immunological synapses, Tcell assays measuring release of effector molecules, especiallyperforin, granzymes or granulolysin or combinations of such T cellassays (see e.g. Current Protocols in Immunology, Current Protocols inCell Biology).

In order to identify the molecular determinants of immune-protectionagainst HCV a specific method of epitope capturing was applied usingsynthetic peptides representing the conserved parts of HCV genotypes 1,2 and 3. Focusing on conserved regions ensures broad applicability ofthe epitopes. Moreover, these regions probably cannot easily be mutatedby the virus, thus minimizing the danger of evolution of immune-escapevariants.

With the methods of the present invention novel HCV-epitopes aredetected. According to a further aspect, the present invention thereforealso provides HCV T cell epitopes identifiable by a method according tothe present invention, said T cell epitopes preferably being selectedfrom the group consisting of polypeptides comprising the peptidesA120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629,1604 according to Table 1 or 2. These peptides are novel ligands for atleast HLA-DRB1*0101, *0401, *0404, *0701 and thus covering at least45-55% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides 1630, C97, 1547, B94-B98, A272-A276 according to Table 1 or 2.These peptides are novel ligands for at least HLA-DRB1*0101, *0401,*0701 and thus covering at least 40-50% of major populations (see Tab.2).

Preferred polypeptides are selected from the group comprising thepeptides B120, B122, C108, C134, C152 according to Table 1 or 2. Thesepeptides are novel ligands for at least HLA-DRB1*0101, *0404, *0701 andthus covering at least 45% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides 1606, 1607, 1577, 1578 according to Table 1 or 2. Thesepeptides are novel ligands for at least HLA-DRB1*0401, *0404, *0701 andthus covering at least 45% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides B50-52, 1623, C130 according to Table 1 or 2. These peptidesare novel ligands for at least HLA-DRB1*0101, *0401, *0404 and thuscovering at least 40% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides 1603, C96 according to Table 1 or 2. These peptides are novelligands for at least HLA-DRB1*0101, *0701 and thus covering at least 40%of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides C191 according to Table 1, being a novel ligand for at leastHLA-DRB1*0401, *0701 and thus covering at least 40% of major populations(see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides A216-A224, A242-A244, C92-C93 according to Table 1 or 2. Thesepeptides are novel ligands for at least HLA-DRB1*0101, *0401 and thuscovering at least 35% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptide A174 according to Table 1 or 2, being a novel ligand for atleast HLA-DRB1*0404, *0701 and thus covering at least 25-30% of majorpopulations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides B32-B38, B100-B102, C135 according to Table 1 or 2. Thesepeptides are novel ligands for at least HLA-DRB1*0101, *0404 and thuscovering at least 20-25% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptide C162 according to Table 1 or 2, being a novel ligand for atleast HLA-DRB1*0401, *0404 and thus covering at least 20-25% of majorpopulations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides 1618, 1622, 1624, 1546, 1556 according to Table 1 or 2. Thesepeptides are novel ligands for at least HLA-DRB1*0701 and thus coveringat least 25% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides A114, B58, B112-B118, B18-B22, C112, C116, C122, C127, C144,C159-C160, C174, 1558, 1581 according to Table 1 or 2. These peptidesare novel ligands for at least HLA-DRB1*0101 and thus covering at least20% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptide C95, being a novel ligand for at least HLA-DRB1*0401 and thuscovering at least 20% of major populations (see Tab. 2).

Preferred polypeptides are selected from the group comprising thepeptides C129, C157-C158, A254-A258, 1605, C109, C161 according to Table1 or 2. These peptides comprising novel ligands for at leastHLA-DRB1*0404 and thus covering at least 5% of major populations (seeTab. 2).

Preferred polypeptides are selected from the group comprising thepeptides 1547, 1555, 1558, 1559, 1560, 1563, 1592, 1604, 1605, 1616,1621, 1623, 1625, 1627, 1630, 1649, 1650, 1651, 1652, 1654, 1655, 1656according to Table 1 or 2, these peptides displaying immunogenicity inHLA-DRB1*0401 transgenic mice (see Example II) and thus representing orcontaining a confirmed HLA class II T-cell epitope binding to at leastHLA-DRB1*0401 (see Tab. 3).

Preferred polypeptides are selected from the group comprising thepeptides 1545, 1552, 1555, 1558, 1559, 1560, 1577, 1592, 1604, 1605,1615, 1617, 1621, 1627, 1631, 1632, 1641, 1647, 1650, 1651, 1652, 1653,1654, 1655 according to Table 1 or 2, these peptides displayingimmunogenicity in HLA-A*0201 transgenic mice (see Example II) and thusrepresenting or containing a confirmed HLA class I T-cell epitopebinding to at least HLA-A*0201 (see Tab. 3).

Preferred polypeptides which are shown to be HLA-B*0702 epitopes withT-cell activating capacity are selected from the group consisting ofpolypeptides 1506, 1526, 1547, 1552, 1553, 1555, 1558, 1562, 1563, 1565,1577, 1578, 1580, 1587, 1592, 1604, 1605, 1621, 1623, 1624, 1627, 1628,1647, 1650, 1651, 1843 with sequence LPRRGPRL (SEQ ID NO:163) (containedin 1506) and 1838 with sequence SPGALVVGVI (SEQ ID NO:164) (contained in1587) as minimal HLA-B*0702 epitopes.

Peptides 1526, 1565, 1631 are also shown to be immunogenic inHLA-DRB1*0401 transgenic mice contain known class II epitopes. Peptides1526, 1553, 1565, 1587, 1623, 1630 are also shown to be immunogenic inHLA-A*0201 transgenic mice contain known A2 epitopes.

Preferred polypeptides are selected from the group comprising thepeptides listed in tables 3, 5 and the bold peptides in 7 (“hotspots”).

The preferred polypeptides mentioned above also include all fragmentscontaining the minimal sequence of the epitope, i.e. the 8- or 9-merbeing necessary for binding to MHC/HLA molecules.

Preferably, the epitopes or peptides according to the present inventionfurther comprises 1 to 30, preferably 2 to 10, especially 2 to 6,naturally occurring amino acid residues at the N-terminus, theC-terminus or at the N- and C-terminus. For the purposes of the presentinvention the term “naturally occurring” amino acid residue relates toamino acid residues present in the naturally occurring protein at thespecific position, relative to the epitope or peptide. For example, forthe HLA-A2 epitope with the amino acid sequence HMWNFISGI (SEQ IDNO:192) contained within peptide ID 1565 (Tab. 1), the naturallyoccurring amino acid residue at the N-terminus is −K; the threenaturally occurring amino acid residues at the C-terminus are −QYL. A“non-naturally occurring” amino acid residue is therefore any amino acidresidue being different as the amino acid residue at the specificposition relative to the epitope or peptide.

According to a preferred embodiment of the present invention, thepresent epitopes or peptides further comprise non-naturally occurringamino acid(s), preferably 1 to 1000, more preferred 2 to 100, especially2 to 20 non-naturally occurring amino acid residues, especially at theN-terminus, the C-terminus or at the N- and C-terminus. Alsocombinations of non-naturally and naturally occurring amino acidresidues are possible under this specific preferred embodiment. Thepresent epitope may also contain modified amino acids (i.e. amino acidresidues being different from the 20 “classical” amino acids, such asD-amino acids or S—S bindings of Cys) as additional amino acid residuesor in replacement of a naturally occurring amino acid residue.

It is clear that also epitopes or peptides derived from the presentepitopes or peptides by amino acid exchanges improving, conserving or atleast not significantly impeding the T cell activating capability of theepitopes are covered by the epitopes or peptides according to thepresent invention. Therefore, the present epitopes or peptides alsocover epitopes or peptides, which do not contain the original sequenceas derived from a specific strain of HCV, but trigger the same orpreferably an improved T cell response. These epitopes are referred toas “heteroclitic”. These include any epitope, which can trigger the sameT cells as the original epitope and has preferably a more potentactivation capacity of T cells preferably in vivo or also in vitro. Alsothe respective homologous epitopes from other strains of HCV areencompassed by the present invention.

Heteroclitic epitopes can be obtained by rational design i.e. takinginto account the contribution of individual residues to binding toMHC/HLA as for instance described by Ramensee et al. 1999 or Sturnioloet al. 1999, combined with a systematic exchange of residues potentiallyinteracting with the TCR and testing the resulting sequences with Tcells directed against the original epitope. Such a design is possiblefor a skilled man in the art without much experimentation.

Another possibility includes the screening of peptide libraries with Tcells directed against the original epitope. A preferred way is thepositional scanning of synthetic peptide libraries. Such approaches havebeen described in detail for instance by Blake et al 1996 and Hemmer etal. 1999 and the references given therein.

As an alternative to epitopes represented by the cognate HCV derivedamino acid sequence or heteroclitic epitopes, also substances mimickingthese epitopes e.g. “peptidemimetica” or “retro-inverso-peptides” can beapplied.

Another aspect of the design of improved epitopes is their formulationor modification with substances increasing their capacity to stimulate Tcells. These include T helper cell epitopes, lipids or liposomes orpreferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes istheir formulation with immune stimulating substances for instancecytokines or chemokines like interleukin-2, -7, -2, -18, class I and IIinterferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligandand others.

According to a further aspect, the present invention is drawn to the useof a HCV epitope or HCV peptide according to the present invention forthe preparation of a HLA restricted vaccine for treating or preventinghepatitis C virus (HCV) infections.

The invention also encompasses the use of an epitope according to thepresent invention for the preparation of a vaccine for treating orpreventing preventing hepatitis C virus (HCV) infections.

Consequently, the present invention also encompasses a vaccine fortreating or preventing hepatitis C virus (HCV) infections comprising anepitope according to the present invention.

Furthermore, also a HLA specific vaccine for treating or preventinghepatitis C virus (HCV) infections comprising the epitopes or peptidesaccording to the present invention is an aspect of the presentinvention.

Preferably, such a vaccine further comprises an immunomodulatingsubstance, preferably selected from the group consisting of polycationicsubstances, especially polycationic polypeptides, immunomodulatingnucleic acids, especially deoxyinosine and/or deoxyuracile containingoligodeoxynucleotides, or mixtures thereof.

Preferably the vaccine further comprises a polycationic polymer,preferably a polycationic peptide, especially polyarginine, polylysineor an antimicrobial peptide.

The polycationic compound(s) to be used according to the presentinvention may be any polycationic compound, which shows thecharacteristic effect according to the WO 97/30721. Preferredpolycationic compounds are selected from basic polypeptides, organicpolycations, basic polyaminoacids or mixtures thereof. Thesepolyaminoacids should have a chain length of at least 4 amino acidresidues. Especially preferred are substances containing peptidicbounds, like polylysine, polyarginine and polypeptides containing morethan 20%, especially more than 50% of basic amino acids in a range ofmore than 8, especially more than 20, amino acid residues or mixturesthereof. Other preferred polycations and their pharmaceuticalcompositions are described in WO 97/30721 (e.g. polyethyleneimine) andWO 99/38528. Preferably these polypeptides contain between 20 and 500amino acid residues, especially between 30 and 200 residues.

These polycationic compounds may be produced chemically or recombinantlyor may be derived from natural sources.

Cationic (poly)peptides may also be polycationic anti-bacterialmicrobial peptides. These (poly)peptides may be of prokaryotic or animalor plant origin or may be produced chemically or recombinantly. Peptidesmay also belong to the class of defensines. Such host defense peptidesor defensines are also a preferred form of the polycationic polymeraccording to the present invention. Generally, a compound allowing as anend product activation (or down-regulation) of the adaptive immunesystem, preferably mediated by APCs (including dendritic cells) is usedas polycationic polymer.

Especially preferred for use as polycationic substance in the presentinvention are cathelicidin derived antimicrobial peptides or derivativesthereof (WO 02/13857), incorporated herein by reference), especiallyantimicrobial peptides derived from mammal cathelicidin, preferably fromhuman, bovine or mouse, or neuroactive compounds, such as (human) growthhormone (as described e.g. in WO01/24822).

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin, especially mouse, bovine or especially humancathelins and/or cathelicidins. Related or derived cathelin substancescontain the whole or parts of the cathelin sequence with at least 15-20amino acid residues. Derivations may include the substitution ormodification of the natural amino acids by amino acids, which are notamong the 20 standard amino acids. Moreover, further cationic residuesmay be introduced into such cathelin molecules. These cathelin moleculesare preferred to be combined with the antigen/vaccine compositionaccording to the present invention. However, these cathelin moleculessurprisingly have turned out to be also effective as an adjuvant for aantigen without the addition of further adjuvants. It is thereforepossible to use such cathelin molecules as efficient adjuvants invaccine formulations with or without further immunactivating substances.

Another preferred polycationic substance to be used according to thepresent invention is a synthetic peptide containing at least 2KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids,especially L (WO 02/32451, incorporated herein by reference).

The immunomodulating (or:immunogenic) nucleic acids to be used accordingto the present invention can be of synthetic, prokaryotic and eukaryoticorigin. In the case of eukaryotic origin, DNA should be derived from,based on the phylogenetic tree, less developed species (e.g. insects,but also others). In a preferred embodiment of the invention theimmunogenic oligodeoxynucleotide (ODN) is a synthetically producedDNA-molecule or mixtures of such molecules. Derivatives or modificationsof ODNs such as thiophosphate substituted analogues (thiophosphateresidues substitute for phosphate) as for example described in USpatents U.S. Pat. No. 5,723,335 and U.S. Pat. No. 5,663,153, and otherderivatives and modifications, which preferably stabilize theimmunostimulatory composition(s) but do not change their immunologicalproperties, are also included. A preferred sequence motif is a six baseDNA motif containing an (unmethylated) CpG dinucleotide flanked by two5′ purines and two 3′ pyrimidines (5′-Pur-Pur-C-G-Pyr-Pyr-3′). The CpGmotifs contained in the ODNs according to the present invention are morecommon in microbial than higher vertebrate DNA and display differencesin the pattern of methylation. Surprisingly, sequences stimulating mouseAPCs are not very efficient for human cells. Preferred palindromic ornon-palindromic ODNs to be used according to the present invention aredisclosed e.g. in Austrian Patent applications A 1973/2000, A 805/2001,EP 0 468 520 A2, WO 96/02555, WO 98/16247, WO 98/18810, WO 98/37919, WO98/40100, WO 98/52581, WO 98/52962, WO 99/51259 and WO 99/56755 allincorporated herein by reference. Apart from stimulating the immunesystem certain ODNs are neutralizing some immune responses. Thesesequences are also included in the current invention, for example forapplications for the treatment of autoimmune diseases. ODNs/DNAs may beproduced chemically or recombinantly or may be derived from naturalsources. Preferred natural sources are insects.

Alternatively, also nucleic acids based on inosine and cytidine (as e.g.described in the WO 01/93903) or deoxynucleic acids containingdeoxy-inosine and/or deoxyuridine residues (described in WO 01/93905 andPCT/EP 02/05448, incorporated herein by reference) may preferably beused as immunostimulatory nucleic acids for the present invention.

Of course, also mixtures of different immunogenic nucleic acids may beused according to the present invention.

Preferably, the present vaccine further comprises a pharmaceuticallyacceptable carrier.

According to a further preferred embodiment, the present vaccinecomprises an epitope or peptide which is provided in a form selectedfrom peptides, peptide analogues, proteins, naked DNA, RNA, viralvectors, virus-like particles, recombinant/chimeric viruses, recombinantbacteria or dendritic cells pulsed with protein/peptide/RNA ortransfected with DNA comprising the epitopes or peptides.

According to a further aspect, the present invention is drawn to Tcells, a T cell clone or a population (preparation) of T cellsspecifically recognizing any HCV epitope or peptide according to thepresent invention, especially a HCV epitope as described above. Apreferred application of such T cells is their expansion in vitro anduse for therapy of patients e.g. by adoptive transfer. Therefore, thepresent invention also provides the use of T cells, a T cell clone or apopulation (preparation) of T cells for the preparation of a compositionfor the therapy of HCV patients.

Such T cells (clones or lines) according to the present invention,specifically those recognizing the aforementioned HCV peptides are alsouseful for identification of heteroclitic epitopes, which are distinctfrom the originally identified epitopes but trigger the same T cells.

Such cells, compositions or vaccines according to the present inventionare administered to the individuals in an effective amount.

According to a further aspect, the present invention also relates to theuse of the peptides with formulae QRKTKRNTN (SEQ ID NO:167), QRKTKRNT(SEQ ID NO:166), or 1615, 1616, 1617 in particular 9meric peptidesderived from the latter 3 peptides with formulae SAKSKFGYG (SEQ IDNO:193), SAKSKYGYG (SEQ ID NO:194), or SARSKYGYG (SEQ ID NO:195) asHLA-B*08 epitopes, especially for the preparation of a pharmaceuticalpreparation for a HLA-B*08 specific vaccine; the use of the peptideswith the formulae RKTKRNTNR (SEQ ID NO:170) as HLA-B*2705 epitope,especially for the preparation of a pharmaceutical preparation for aHLA-B*2705 specific vaccine; and the use of the peptides with theformulae ARLIVFPDL (SEQ ID NO:1053) as HLA-B*2705 and HLA-B*2709specific vaccine. Further, it also relates to the use of the hotspotepitopes selected from the group of peptides 1835, 84EX, 87EX, 89EX,1426, 1650, 1836, 1846, 1651, 1800, 1799, C114, 1827, C112, C114EX,1827EX, 1798, 1604, 1829, 1579, 1624, 1848, 1547, A1A7, A122EX, A122,1825, A241, B8B38, C70EX, C92, C97, C106, and C134 according to table 7for the preparation of a vaccine comprising synthetic peptides,recombinant protein and/or DNA constitutes of such epitopes.

In particular, two or more epitope hotspots can be combined, with orwithout linker sequences. Preferred linker sequences consist forinstance of 3 to 5 glycine, or alanine or lysine residues. This may beachieved by peptide synthesis, However, combination of hotspots mayresult in quite long polypeptides. In this case, cloning DNA encodingfor such constructs and expressing and purifying the correspondingrecombinant protein is an alternative. Such recombinant proteins can beused as antigens, which in combination with the right adjuvant (IC31,pR, . . . ) can elicit T-cell responses against all the epitopes theyharbor. At the same time, such artificial polypeptides are devoid of theactivities (enzymatic, toxic, immuno-suppressive, . . . ), the naturalHCV antigens may possess.

There are several other ways of delivering T-cell epitope hotspots orcombinations thereof. These include: recombinant viral vectors likevaccinia virus, canary pox virus, adenovirus; self-replicating RNAvectors; “naked DNA” vaccination with plasmids encoding the hotspots orcombination thereof; recombinant bacteria (e.g. Salmonella); dendriticcells pulsed with synthetic peptides, or recombinant protein, or RNA ortransfected with DNA, each encoding T-cell epitope hotspots orcombinations thereof.

The invention will be explained in more detail by way of the followingexamples and drawing figures, to which, however it is not limited.

FIG. 1 shows 40 peptide mixtures each containing up to 20 HCV derived15- to 23mer peptides.

FIG. 2 shows the Epitope Capture approach using peptide pools and emptyDRB1*0401 molecules.

FIG. 3 shows the Epitope Capture approach using peptide pools and emptyDRB1*0404 molecules.

FIG. 4 shows binding of individual peptides to DRB1*0401.

FIG. 5 shows binding of individual peptides to DRB1*0404.

FIG. 6 shows binding of individual peptides to DRB1*0101.

FIG. 7 shows peptides binding to DRB1*0701.

FIG. 8 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+or CD4+ cells from HLA-DRB1*0401 tg mice vaccinated with Ipep1604+IC31.

FIG. 9 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+or CD4+ cells from HLA-A*0201 tg mice vaccinated with Ipep1604+IC31.

FIG. 10 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+or CD4+ cells from HLA-B*0702 tg mice vaccinated with Ipep1604+IC31.

EXAMPLES

General description of the examples:

The present examples show the performance of the present invention on aspecific pathogen hepatitis C virus (HCV).

In the first part the method according to the present invention wasapplied, which is based on the use of “empty HLA molecules”. Thesemolecules were incubated with mixtures of potential HCV derived peptideligands, screening for specific binding events. The possibility to usehighly complex mixtures allows a very quick identification of the fewbinders out of hundreds or even thousands of potential ligands. This isdemonstrated by using HLA-DRB1*0101, -DRB1*0401, -DRB1*0404, -DRB1*0701molecules and pools of overlapping 15- to 23mers. Importantly, thisanalysis using multiple different HLA-alleles allows identifyingpromiscuous ligands capable to binding to more than one HLA allele.Promiscuous T-cell epitopes are particularly valuable components ofepitope-based vaccines. They enable treating a higher portion of apopulation than epitopes restricted to one HLA allele.

The same process can be applied for class I molecules and peptides ofappropriate length i.e. 8 to 11-mers. The ligand-pools can be syntheticoverlapping peptides. Another possibility is to digest the antigen inquestion enzymatically or non-enzymatically. The latter achieved byalkali-hydrolysis generates all potential degradation products and hasbeen successfully used to identify T cell epitopes (Gavin 1993).Enzymatic digestions can be done with proteases. One rational way wouldfurther be to use proteases involved in the natural antigen-processingpathway like the proteasome for class I restricted epitopes (Heemels1995) or cathepsins for class II restricted epitopes (Villadangos 2000).Ligand pools could also be composed of naturally occurring ligandsobtained for instance by lysis of or elution from cells carrying therespective epitope. In this regard it is important to note that alsonon-peptide ligands like for instance glycolipids can be applied. It isknown that nonclassical class I molecules, which can be encoded by theMHC (e.g. HLA-G, HLA-E, MICA, MICB) or outside the MHC (e.g. CD1 family)can present various non-peptide ligands to lymphocytes (Kronenberg1999). Use of recombinant “empty” nonclassical class I molecules wouldallow binding reactions and identification of binders in similar manneras described here.

After rapid identification of ligands capable of binding to HLAmolecules the process according to the present invention also offersways to characterize directly specific T cell responses against thesebinders. One possibility is to directly use the isolated HLA:ligandcomplex in a so called “synthetic T cell assay”. The latter involvesantigen-specific re-stimulation of T cells by the HLA:ligand complextogether with a second signal providing co-stimulation like activationof CD28 by an activating antibody. This assay can be done in an ELIspotreadout.

Another possibility is the immunization of HLA-transgenic mice to proveimmunogenicity of ligands identified by the Epitope Capture approach asdemonstrated in Example II.

Materials & Methods

Peptides

In order to identify conserved regions between HCV genotypes 1, 2 and 3,about 90 full genomes publicly available through Genebank were aligned.In total, 43% of the coding region of HCV was found to be conserved inat least 80% of clinical isolates. In cases, where at a certain positionconsistently two distinct amino acids (eg. arginine or lysine) werefound, both variants were considered for analysis. Altogether 148conserved regions, longer than 8 amino acids were identified. Conservedregion were spanned by ˜500 fifteen amino acid residue (15mer) peptides,each peptide overlapping its precursor by 14 out of 15 amino acids.Conserved regions between 8 and 14 amino acids long were covered byfurther 80 (non-overlapping) 15mers. 15mers were synthesized usingstandard F-moc chemistry in parallel (288 at a time) on a Syro IIsynthesizer (Multisyntech, Witten, Germany). Each fourth 15mer waschecked by mass spectrometry. 15mers were applied for experimentswithout further purification. In addition 63 peptides of 16-xx aa weresynthesized using standard F-moc chemistry on an ABI 433A synthesizer(Applied Biosystems, Weiterstadt, Germany) and purified by RP-HPLC(Biocut 700E, Applied Biosystems, Langen, Germany) using a C18 column(either ODS ACU from YMC or 218TP, Vydac). Purity and identity werecharacterized by MALDI-TOF on a Reflex III mass-spectrometer (Bruker,Bremen, Germany). Peptides were solubilized in 100% DMSO at ˜10 mg/ml(˜5 mM). Stocks of peptide pools (20 peptides each) were made in 100%DMSO at a final concentration of 0.5 mg/ml (˜0.25 mM) for each peptide.All peptides used in the present invention are listed in Table 1.Peptides YAR (YARFQSQTTLKQKT (SEQ ID NO:196)), HA (PKYVKQNTLKLAT (SEQ IDNO:197)), P1 (GYKVLVLNPSVAAT (SEQ ID NO:198)),P2(HMWNFISGIQYLAGLSTLPGNPA (SEQ ID NO:199)), P3(KFPGGGQIVGVYLLPRRRGPRL(SEQ ID NO:200)), P4 (DLMGYIPAV (SEQ ID NO:201)) and CLIP(KLPKPPKPVSKMRMATPLLMQALPM (SEQ ID NO:202)) were used as controlpeptides in binding assays.

Epitope Capture and Peptide Binding Assay

Soluble HLA class II DRA1*0101/DRB1*0101/Ii, DRA1*0101/DRB1*0401/Ii,DRA1*0101/DRB1*0404/Ii and DRA1*0101/DRB1*0701/Ii molecules wereexpressed in SC-2 cells and purified as described in Aichinger et al.,1997. In peptide binding reactions soluble DRB1*0101, DRB1*0401,DRB1*0404 molecules were used in a concentration of ˜0.5 μM, and eachsingle peptide was added in 10-fold molar excess (5 μM) if not mentioneddifferently. The concentration of DMSO in the binding reaction did notexceed 4%. The reaction was performed in PBS buffer (pH 7.4) at roomtemperature for 48 hours in the presence of a protease inhibitorcocktail (Roche) and 0.1% octyl-beta-D-glucopyranoside (Sigma). Peptidebinding was evaluated in an SDS-stability assay (Gorga et al., 1987):trimeric HLA class II alpha:beta:peptide complexes are resistant to SDSand consequently appear as ˜60 kDa band in SDS-PAGE Western blotanalysis. Individual HLA class II alpha- and beta-chains not stabilizedby bound peptide migrate as ˜35 kDa and ˜25 kDa bands, respectively.Briefly, HLA-peptide complexes were treated with 1% SDS at roomtemperature and resolved by SDS-PAGE run with 20 mA for approximately2.5 hours at room temperature. Protein was transferred onto PVDFmembrane by electroblotting, and stained with anti-alpha-chain TAL.1B5or/and beta-chain MEM136 antibodies. For detection of Western-blotsignals ECL solutions (Amersham) were used. For DRB1*0101 molecules HAand P1 peptides were used as controls for evaluation of strong binding,P2 peptide for intermediate binding and YAR as a negative control. ForDRB1*0401 the strongest binding controls were YAR and HA peptides, whileP1 and P2 served as an intermediate and weak binder, respectively. Inthe case of DRB1*0404 molecules P1 and P2 peptides were used to estimatestrong binding, YAR peptide to control intermediate binding and HApeptide as an negative control. The binding affinities to DRB1*0701 weretest by a peptide-competition assay (Reay et al., 1992). Briefly,binding of the biotinylated CLIP peptide with high affinity (referencepeptide) has been used for monitoring of HLA:peptide complex formation.A testing peptide added to the binding reaction at an equimolarconcentration to CLIP peptide could compete out CLIP when its affinityis higher or inhibit binding for 50% if its affinity is equal toaffinity of CLIP. In the case of lower affinity peptides they should beadded in excess to the reference peptide to compete for occupancy of HLAbinding grove. The values of the concentration of competitor peptidesrequired for 50% inhibition of reference peptide (biotinylated CLIP)binding (IC₅₀) can be used for evaluation of peptide binding affinities.Alternatively, comparing of the amount of reference peptide bound to HLAmolecules in the presence or absence of competitor peptide one candetermine the binding activity of the peptide of interest. In thepresent peptide-competition assay conditions of peptide binding weresimilar to described above. DRB1*0701 molecules were used in aconcentration of ˜0.5 μM and biotinylated CLIP was added to all samplesin the final concentration of 2 μM. Competitor peptides were added inthree different concentrations: 2 nM, 20 μM and 200 μM. Binding reactionwas performed in PBS buffer (pH 7.4) for 18 hours at 37° C. The amountof biotinylated CLIP associated with soluble DRB1*0701 molecules wasdetermined by ELISA. Briefly, MaxiSorp 96-well plates (Nunc, Denmark)were coated with mouse anti-DR antibody L243 by overnight incubationwith 50 μl of 10 μg/ml dilution in PBS at 4° C. Non-specific binding towells a was blocked by incubation with T-PBS containing 3% of BSA for 2hours at 37° C. and binding reactions were then “captured” for 2 hoursat room temperature. Following extensive washing, HLA-assosiated peptidecomplexes were detected using alkaline phosphatase-streptavidin (Dako)and Sigma 104 phosphatase substrate. A microplate reader (VICTOR) wasused to monitor optical density at 405 nm. Non-biotinylated CLIP, P1 andP2 peptides were used as positive controls to evaluate strong binding.Peptide P3 and P4 served as a weakly binding and non-binding control,respectively.

Immunization of HLA-Transgenic Mice

Immunogenicity of synthetic HCV-derived peptides was tested inHLA-DRB1*0401- and HLA-A*0201-transgenic mice as follows: Groups of 3mice (female, 8 weeks of age) were injected subcutaneously into theflank (in total 100 μg of peptide +30 μg oligodinucleotide CpI (Purimex,Göttingen, Germany) per mouse). One week after the vaccination, spleenswere removed and the splenocytes were activated ex vivo with the peptideused for vaccination and an irrelevant negative control peptide todetermine IFN-gamma-producing specific cells (mouse ELISpot assay).

Mouse splenocyte ELIspot assay for single cell IFN-gamma release ELISpotplates (MAHA S4510, Millipore, Germany) were rinsed with PBS (200μl/well), coated with anti-mouse IFN-gamma mAb (clone R46A2; 100 μl/wellof 5 μg/ml in 0.1 M NaHCO₃, pH 9.2-9.5) and incubated overnight at 4° C.Plates were washed four times with PBS/0.1% Tween 20 and incubated withPBS/1% BSA (200 μl/well) at room temperature for 2 h to blocknonspecific binding. Spleen cells from vaccinated mice were prepared andplated at 1×10⁶-3×10⁵ cells/well and incubated overnight at 37° C./5%CO₂ either in the presence of the immunizing antigen (peptide), controlpeptides or with medium alone. Subsequently, plates were washed fourtimes and incubated with biotinylated anti-mouse IFN-gamma mAb (cloneAN18.17.24, 100 μl/well of 2 μg/ml in PBS/1% BSA) for 2 h at 37° C.After washing, streptavidin-peroxidase (Roche Diagnostics, Vienna,Austria) was added ( 1/5000 in PBS, 100 μl/well) and plates wereincubated at room temperature for 2 additional hours. Subsequently,substrate was added to the washed plates (100 μl/well of a mixture of 10ml 100 mM Tris pH 7.5 supplemented with 200 μl of 40 mg/ml DAB stockcontaining 50 μl of 80 mg/ml NiCl₂ stock and 5 μl of 30% H₂O₂). Thereaction was stopped after 20-30 minutes by washing the plates with tapwater. Dried plates were evaluated with an ELISpot reader (BIOREADER2000, BioSys, Karben, Germany).

IFN-Gamma ELIspot with Human PBMC

PBMC from HCV RNA-negativ therapy responders or subjects spontaneouslyrecovered were collected and HLA-typed serologically. Whole blood wascollected in ACD Vacutainer tubes (Becton Dickinson Europe, Erembodegem,Germany). PBMC were isolated on Lymphoprep (Nycomed Pharma AS, Oslo,Norway) using Leuco-sep tubes (Greiner, Frickenhausen, Germany), washed3× with PBS (Invitrogen Life Technologies (formerly GIBCOBRL), Carlsbad,Calif., USA) and resuspended at a concentration of 2×10⁷/ml in freezingmedium consisting of 4 parts RPMI 1640 supplemented with 1 mM sodiumpyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 μM2-mercaptoethanol (all from Invitrogen Life Technologies), 9 partsfoetal bovine serum (FCS; from PAA, Linz, Austria) and 1 part DMSO(SIGMA, Deisenhofen, Germany). PBMC were stored over night in 1° C.freezing containers (Nalgene Nunc International, Rochester, N.Y., USA)at −80° C. and then transferred into liquid nitrogen. The ELIspot assaywas essentially done as described (Lalvani et al.). Briefly, MultiScreen 96-well filtration plates MAIP S4510 (Millipore, Bedford, Mass.)were coated with 10 μg/ml (0,75 μg/well) anti-human IFN-g monoclonalantibody (Mab) B140 (Bender Med Systems, Vienna, Austria) over night at4° C. Plates were washed 2 times with PBS (Invitrogen Life Technologies)and blocked with ELIspot medium (RPMI 1640 supplemented with 1 mM sodiumpyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 μM2-mercaptoethanol (all from Invitrogen Life Technologies) and 10% humanserum type AB (PAA, Linz, Austria). Cryo-preserved PBMC were thawedquickly in a 37° C. water bath, washed 1× with ELISPOT medium andincubated overnight (37° C., 5% CO₂). The next day cells were plated at200,000 PBMC/well and co-cultivated with either individual peptides (10μg/ml) or peptide pools (each peptide at a final concentration of 5μg/ml) for 20 hrs. After removing cells and washing 6 times with washbuffer (PBS; 0,1% Tween 20 from SIGMA), 100 μl of a 1:10000 dilution(0.015 μg/well) of the biotinylated anti-human IFN-γ MAb B308-BT2(Bender Med Systems), was added for an incubation of 2 hrs at 37° C. oralternatively for over night at 4° C. After washing,Streptavidin-alkaline phosphatase (DAKO, Glostrup, Denmark) was added at1.2 μg/ml for 1 hr at 37° C. The assay was developed by addition of 100μl/well BCIP/NBT alkaline phosphatase substrate (SIGMA).

In Vitro Priming of Human PBMCs

Human PBMCs are repeatedly stimulated with antigen (peptide or peptidemixture) in the presence of IL-2 and IL-7. This leads to the selectiveoligoclonal expansion of antigen-specific T cells. Responses againstindividual epitopes can be assessed for instance by IFN-γ ELIspotassays. Freshly thawed PBMCs were cultured in 6 well plates (2-4×10⁶/mLviable cells) in RPMI-1640 (GibcoBRL), 1% non-essential amino acids(GibcoBRL, cat# 11140-035), 1% Penicillin (10,000 U/ml)-Streptomycin(10,000 μg/ml) (GibcoBRL, cat#15140-122), 1% L-Glutamine (GibcoBRL),0.1% beta-mercapto-ethanol (GibcoBRL), 1% Na-pyruvate (GibcoBRL), plus10% Human AB serum (PAA, Linz, Austria). Peptides (10 μM each) wereadded to each well. rhIL-7 (Strathmann Biotech) was added at 10 ng/mLfinal concentration. 20-30 U/mL rhIL-2 (Strathmann Biotech) were addedon day 4. On day 10, all cells were removed from plates, washed once inmedia (as above), and counted. For the next cycle of in vitro priming,viable cells were co-cultivated with autologous gamma irradiated (1.2gray/min, for 20 minutes) PBMC as feeders (plated at 100,000 per well)and peptides, rh-IL-2 as described above. ELIspot was done as describedabove, except that 200,000 responder cells (pre-stimulated for 2 roundsof in vitro priming) were used together with 60,000 autologousirradiated responder cells.

Example I Rapid Identification of Promiscuous HLA-Binding Peptides fromHcv by Measuring Peptide Pools Arrayed in Matrix Format

To span conserved regions within the HCV polyprotein more than 640peptides were synthesized (Table 1). For rapid identification of HLAligands and novel T-cell epitopes, 40 peptide pools each containing 20single peptides were prepared. The pools were constructed in a way thateach peptide was present in 2 pools (matrix format). This allowsidentification of reactive peptides at the crossover points of row- andcolumn mixtures (FIG. 1 HCV peptide matrix).

TABLE 1 Synthetic peptides derived from conserved regions of HCV PEPTIDEID SEQ ID PEPTIDE A1 203 MSTNPKPQRKTKRNT A2 204 STNPKPQRKTKRNTN A3 205TNPKPQRKTKRNTNR A4 206 NPKPQRKTKRNTNRR A5 207 PKPQRKTKRNTNRRP A6 208KPQRKTKRNTNRRPQ A7 209 PQRKTKRNTNRRPQD A8 210 QRKTKRNTNRRPQDV A9 211RKTKRNTNRRPQDVK A10 212 KTKRNTNRRPQDVKF A11 213 TKRNTNRRPQDVKFP A12 214KRNTNRRPQDVKFPG A13 215 RNTNRRPQDVKFPGG A14 216 NTNRRPQDVKFPGGG A15 217TNRRPQDVKFPGGGQ A16 218 NRRPQDVKFPGGGQI A17 219 RRPQDVKFPGGGQIV A18 220RPQDVKFPGGGQIVG A19 221 PQDVKFPGGGQIVGG A20 222 QDVKFPGGGQIVGGV A21 223DVKFPGGGQIVGGVY A22 224 VKFPGGGQIVGGVYL A23 225 KFPGGGQIVGGVYLL A24 226FPGGGQIVGGVYLLP A25 227 PGGGQIVGGVYLLPR A26 228 GGGQIVGGVYLLPRR A27 229GGQIVGGVYLLPRRG A28 230 GQIVGGVYLLPRRGP A29 231 QIVGGVYLLPRRGPR A30 232IVGGVYLLPRRGPRL A31 233 VGGVYLLPRRGPRLG A32 234 GGVYLLPRRGPRLGV A33 235GVYLLPRRGPRLGVR A34 236 VYLLPRRGPRLGVRA A35 237 YLPRRGPRLGVRAT A36 238LLPRRGPRLGVRATR A37 239 LPRRGPRLGVRATRK A38 240 PRRGPRLGVRATRKT A39 241RRGPRLGVRATRKTS A40 242 RGPRLGVRATRKTSE A41 243 GPPWGVRATRKTSER A42 244PRLGVRATRKTSERS A43 245 RLGVRATRKTSERSQ A44 246 LGVRATRKTSERSQP A45 247GVRATRKTSERSQPR A46 248 VRATRKTSERSQPRG A47 249 RATRKTSERSQPRGR A48 250ATRKTSERSQPRGRR A49 251 TRKTSERSQPRGRRQ A50 252 RKTSERSQPRGRRQP A51 253KTSERSQPRGRRQPI A52 254 TSERSQPRGRRQPIP A53 255 SERSQPRGRRQPIPK A54 256DPRRRSRNLGKVIDT A55 257 PRRRSRNLGKVIDTL A56 258 RRRSRNLGKVIDTLT A57 259RRSRNLGKVIDTLTC A58 260 RSRNLGKVIDTLTCG A59 261 SRNLGKVIDTLTCGF A60 262RNLGKVIDTLTCGFA A61 263 NLGKVIDTLTCGFAD A62 264 LGKVIDTLTCGFADL A63 265GKVIDTLTCGFADLM A64 266 KVIDTLTCGFADLMG A65 267 VIDTLTCGFADLMGY A66 268IDTLTCGFADLMGYI A67 269 DTLTCGFADLMGYIP A68 270 TLTCCFADLMGYIPL A69 271LTCGFADLMGYIPLV A70 272 TCGFADLMGYIPLVG A71 273 CGFADLMGYIPLVGA A72 274GFADLMGYIPLVGAP A73 275 FADLMGYIPLVGAPL A74 276 ADLMGYIPLVGAPLG A75 277DLMGYIPLVGAPLGG A76 278 DPRHRSRNVGKVIDT A77 279 PRHRSRNVGKVIDTL A78 280RHRSRNVGKIDTLT B41 281 CECYDAGAAWYELTP B42 282 ECYDAGAAWYELTPA B43 283CYDAGAAWYELTPAE B44 284 YDAGAAWYELTPAET B45 285 DAGAAWYELTPAETT B46 286AGAAWYELTPAETTV B47 287 GAAWYELTIPAETTV B48 288 AAWYELTPAETTVRL B49 289DAGAAWYELTPAETS B50 290 AGAAWYELTPAETSV B51 291 GAAWYELTPAETSVR B52 292AAWYELTPAETSVRL B53 293 FWAKHMWNFISGIQY B54 294 WAKHMWNFISGIQYL B55 295AKHMWNFISGIQYLA B56 296 KHMWNFISGIQYLAG B57 297 HMWNFISGIQYLAGL B58 298MWNFISGIQYLAGLS B59 299 WNFISGIQYLAGLST B60 300 NFISGIQYLAGLSTL B61 301FISGIQYLAGLSTLP B62 302 ISGIQYLAGLSTLPG B63 303 SGIQYLAGLSTLPGN B64 304GIQYLAGLSTLPGNP B65 305 IQYLAGLSTLPGNPA B66 306 QYLAGLSTLPGNPAI B67 307YLAGLSTLPGNPAIA B68 308 LAGLSTLPGNPAIAS B69 309 AGLSTLPGNPAIASI B70 310GLSTLPGNPAIASLM B71 311 LSTLPGNPAIASLMA B72 312 STLPGNPAIASLMAF B73 313QYLAGLSTLPGNPAV B74 314 YLAGLSTLPGNPAVA B75 315 LAGLSTLPGNPAVAS B76 316AGLSTLPGNPAVASM B77 317 GLSTLPGNPAVASMM B78 318 LSTLPGNPAVASMMA B79 319STLPGNPAVASMMAF B80 320 GAAVGSIGLGKVLVD B81 321 AAVGSIGLGKVLVDI B82 322AVGSIGLGKVLDIL B83 323 VGSIGLGKVLVDILA B84 324 GSIGLGKVLVDILAG B85 325SIGLGKVLVDILAGY B86 326 IGLGKVLVDILAGYG B87 327 GLGKVLVDILAGYGA B88 328LGKVLVDILAGYGAG B89 329 GKVLVDILAGYGAGV B90 330 KVLVDILAGYGAGVA B91 331VLVDILAGYGAGVAG B92 332 LVDILAGYGAGVAGA B93 333 VDILAGYGAGVAGAL B94 334DILAGYGAGVAGALV B95 335 ILAGYGAGVAGALVA B96 336 LAGYGAGVAGALVAF B97 337AGYGAGVAGALVAFK B98 338 GYGAGVAGALVAFKI B99 339 YGAGVAGALVAFKIM B100 340GAGVAGALVAFKIMS B101 341 AGVAGALVAFKIMSG B102 342 GVAGALVAFKIMSGE B103343 GYGAGVAGALVAFKV B104 344 YGAGVAGALVAFKVM B105 345 GAGVAGALVAFKVMSB106 346 AGVAGALVAFKVMSG B107 347 GVAGALVAFKVMSGE B108 348GKVLVDILAGYGAGI B109 349 KVLVDILAGYGAGIS B110 350 VLVDILAGYGAGISG B111351 LVDILAGYGAGISGA B112 352 VDILAGYGAGISGAL B113 353 DILAGYGAGISGALVB114 354 ILAGYGAGISGALVA B115 355 LAGYGAGISGALVAF B116 356AGYGAGISGALVAFK B117 357 GYGAGISGALVAFKI B118 358 YGAGISGALVAFKIM B119359 GAGISGALVAFKIMS A79 360 HRSRNVGKVIDTLTC A80 361 RSRNVGKVIDTLTCG A81362 SRNVGKVIDTLTCGF A82 363 RNVGKVIDTLTCGFA A83 364 NVGKVIDTLTCGFAD A84365 VGKVIDTLTCGFADL A85 366 TLTCGFADLMGYIPV A86 367 LTCGFADLMGYIPVV A87368 TCGFADLMGYIPVVG A88 369 CGFADLMGYIPVVGA A89 370 GFADLMGYIPVVGAP A90371 FADLMGYIPVVGAPL A91 372 ADLMGYIPVVCAPLG A92 373 DLMGYIPVVGAPLGG A93374 ARALAHGVRVLEDGV A94 375 RALAHGVRVLEDGVN A95 376 ALAHGVRVLEDGVNY A96377 LAHGVRVLEDGVNYA A97 378 AHGVRVLEDGVNYAT A98 379 HGVRVLEDGVNYATG A99380 GVRVLEDGVNYATGN A100 381 VRVLEDGVNYATGNL A101 382 RVLEDGVNYATGNLPA102 383 VLEDGVNYATGNLPG A103 384 LEDGGVNYATGNLPGC A104 385EDGVNYATGNLPGCS A105 386 DGVNYATGNLPGCSF A106 387 GVNYATGNLPGCSES A107388 VNYATGNLPGCSFSI A108 389 NYATGNLPGCSFSIF A109 390 YATGNLPGCSFSIFLA110 391 ATGNLPGCSFSIFLL A111 392 TGNLPGCSFSIFLLA A112 393GNLPGCSFSIFLLAL A113 394 NLPGCSFSIFLLALL A114 395 LPGCSFSIFLLALLS A115396 PGCSFSIFLLALLSC A116 397 IQLINTNGSWHINRT A117 398 QLINTNGSWHINRTAA118 399 LINTNGSWHINRTAL A119 400 INTNGSWINRTALN A120 401NTNGSWHINRTALNC A121 402 TNGSWHINRTALNCN A122 403 NGSWHINRTALNCND A123404 GSWHINRTALNCNDS A124 405 SWHINRTALNCNDSL A125 406 IQLVNTNGSWHINRTA126 407 QLVNTNGSWHINRTA A127 408 LVNTNGSWHINRTAL A128 409VNTNGSWHINRTALN A129 410 VDYPYRLWHYPCTVN A130 411 DYPYRLWHYPCTVNF A131412 YPYRLWHYPCTVNFT A132 413 PYRLWHYPCTVNFTI A133 414 YRLWHYPCTVNFTIFA134 415 RLWHYPCTVNFTIFK A135 416 LWHYPCTVNFTIFKV A136 417WHYPCTVNFTIFKVR A137 418 HYPCTVNFTIFKVRM A138 419 YPCTVNFTIFKVRMY A139420 PCTVNFTIFKVRMYV A140 421 CTVNFTIFKVRMYVG A141 422 TVNFTIFKVRMYVGGA142 423 VNFTIFKVRMYVGGV A143 424 NFTIFKVRMYVGGVE A144 425FTIFKVRMYVGGVEH A145 426 TIFKVRMYVGGVEHR A146 427 IFKVRMYVGGVEHRL A147428 DYPYRLWHYPCTVNY A148 429 YPYRLWHYPCTVNYT A149 430 PYRLWHYPCTVNYTIA150 431 YRLWHYPCTVNYTIF A151 432 RLWHYPCTVNYTIFK A152 433LWHYPCTVNYTIFKI A153 434 WHYPCTVNYTIFKIR A154 435 HYPCTVNYTIFKIRM A155436 YPCTVNYTIFKIRMY A156 437 PCTVNYTIFKIRMYV B120 438 AGISGALVAFKIMSGB121 439 GISGALVAFKIMSGE B122 440 VNLLPAILSPGALVV B123 441NLLPAILSPGALWG B124 442 LLPAILSPGALVVGV C1 443 LPAILSPGALVVGVV C2 444PAILSPGALVVGVVC C3 445 AILSPGALVVGVVCA C4 446 ILSPGALVVGVVCAA C5 447LSPGALVVGVVCAAI C6 448 SPGALVVGVVCAAIL C7 449 PGALVVGVVCAAILR C8 450GALVVGVV0AAILRR C9 451 ALVVGVVCAAILRRH C10 452 LVVGWCAAILRRHV C11 453VVGVVCAAILRRHVG C12 454 VGVVCAAILRRHVGP C13 455 GVVCAAILRRHVGPG C14 456VVCAAILRRHVGPGE C15 457 VCAAILRRHVGPGEG C16 458 CAAILRRHVGPGEGA C17 459AAILRRHVGPGEGAV C18 460 AILRRHVGPGEGAVQ C19 461 ILRRHVGPGEGAVQW C20 462LRRHVGPGEGAVQWM C21 463 RRHVGPGEGAVQWMN C22 464 RHVGPGEGAVQWMNR C23 465HVGPGEGAVQWMNRL C24 466 VGPGEGAVQWMNRLI C25 467 GPGEGAVQWMNRLIA C26 468PGEGAVQWMNRLIAF C27 469 GEGAVQWMNRLIAFA C28 470 EGAVQWMNRLIAFAS C29 471GAVQWMNRLIAFASR C30 472 AVQWMNRLIAFASRG C31 473 VQWMNRLIAFASRNGN C32 474QWMNRLIAFASRGNH C33 475 WMNRLIAFASRGNHV C34 476 MNRLAFASRGNHVS C35 477NRLIAFASRGNHVSP C36 478 RLIAFASRGNHVSPT C37 479 LIAFASRGNHVSPTH C38 480IAFASRGNHVSPTHY C39 481 AFASRGNHVSPTHYV C40 482 VNLLPGILSPGALVV C41 483NLLPGILSPGALVVG C42 484 LLPGILSPGALVVGV C43 485 LPGILSPGALVVGVI C44 486PGILSPGALVVGVIC C45 487 GILSPGALVVGVICA C46 488 ILSPGALVVGVICAA C47 489LSPGALVVGVICAAI C48 490 SPGALVVGVICAAIL C49 491 PGALVVGVICAAILR C50 492GALVVGVICAAILRR C51 493 ALVVGVICAAILRRH C52 494 LVVGVICAAILRRHV C53 495VVGVI0AAILRRHVG C54 496 VGVIOAAILRRHVGP C55 497 GVICAAILRRHVGPG C56 498VICAAILRRHVGPGE C57 499 CAAILRRHVGPGEG C58 500 MNRLIAFASRGNHVA C59 501NRLIAFASRGNHVAP C60 502 RLIAFASRGNHVAPT C61 503 LIAFASRGNHVAPTH C62 504IAFASRGNHVAPTHY C63 505 AFASRGNHVAPTHYV C64 506 KGGRKPARLIVFPDL C65 507GGRKPARLIVFPDLG C66 508 GRKPARLIVFPDLGV C67 509 RKPARLIVFPDLGVR C68 510KPARLIVFPDLGVRV C69 511 PARLIVFPDLGVRVC C70 512 ARLIVFPDLGVRVCE C71 513RLIVFPDLGVRVCEK C72 514 LIVFPDLGVRVCEKM C73 515 IVFPDLGVRVCEKMA C74 516VFPDLGVRVCEKMAL A157 517 CTVNYTIFKIRMYVG A158 518 TVNYTIFKIRMYVGG A159519 VNYTIFKIRMYVGGV A160 520 NYTIFKIRMYVGGVE A161 521 YTIFKIRMYVGGVEHA162 522 TIFKIRMYVGGVEHR A163 523 IFKIRMYVGGVEHRL A164 524LPALSTGLIHLHQNI A165 525 PALSTGIHLHQNIV A166 526 ALSTGLIHLHQNIVD A167527 LSTGIHLHQNIVDV A165 528 STGLIHLHQNIVDVQ A169 529 TQLIHLHQNIVDVQYA170 530 GLIHLHQNIVDVQYL A171 531 LIHLHQNIVDVQYLY A172 532IHLHQNIVDVQYLYG A173 533 LPALSTGLLHLHQNI A174 534 PALSTGLLHLHQNIV A175535 ALSTGLLHLHQNIVD A176 536 LSTGLLHLHQNIVDV A177 537 STGLLHLHQNIVDVQA178 538 TGLLHLHQNIVDVQY A179 539 GLLHLHQNIVDVQYM A180 540LLHLHQNIVDVQYMY A181 541 LHLHQNIVDVQYMYG A182 542 HLHAPTGSGKSTKVP A183543 LHAPTGSGKSTKVPA A184 544 HAPTGSGKSTKVPAA A185 545 APTGSGKSTKVPAAYA186 546 PTGSGKSTKVPAAYA A187 547 TGSGKSTKVPAAYAA A188 548GSGKSTKVPAAYAAQ A189 549 SGKSTKVPAAYAAQG A190 550 GKSTKVPAAYAAQGY A191551 KSTKVPAAYAAQGYK A192 552 STKVPAAYAAQGYKV A193 553 TKVPAAYAAQGYKVLA194 554 KVPAAYAAQGYKVLV A195 555 VPAAYAAQGYKVLVL A196 556PAAYAAQGYKVLVLN A197 557 AAYAAQGYKVLVLNP A198 558 AYAAQGYKVLVLNPS A199559 YAAQGYKVLVLNPSV A200 560 AAQGYKVLVLNPSVA A201 561 AQGYKVLVLNPSVAAA202 562 QGYKVLVLNPSVAAT A203 563 GYKVLVLNPSVAATL A204 564YKVLVLNPSVAATLG A205 565 KVLVLNPSVAATLGF A206 566 VLVLNPSVAATLGFG A207567 LVLNPSVAATLGFGA A208 568 VLNPSVAATLGFGAY A209 569 YLHAPTGSGKSTKVPA210 570 LHAPTGSGKSTKVPV A211 571 HAPTGSGKSTKVPVA A212 572APTGSGKSTKVPVAY A213 573 PTGSGKSTKVPVAYA A214 574 TGSGKSTKVPVAYAA A215575 GSGKSTKVPVAYAAQ A216 576 SGKSTKVPVAYAAQG A217 577 GKSTKVPVAYAAQGYA218 578 KSTKVPVAYAAQGYK A219 579 STKVPVAYAAQGTKV A220 580TKVPVAYAAQGYKVL A221 581 KVPVAYAAQGYKVLV A222 582 VPVAYAAQGYKVLVL A223583 PVAYAAQGYKVLVLN A224 584 VAYAAQGYKVLVLNP A225 585 ITSTYGKFLADGGCA226 586 TYSTYGKFLADGGCS A227 587 YSTYGKFLADGCCSG A228 588STYGKFLADGGCSGG A229 589 TYGKFLADGGCSGGA A230 590 YGKFLADGGCSGGAY A231591 GKFLADGGCSGGAYD A232 592 KFLADGGCSGGAYDI A233 593 FLADGGCSGGAYDIIA234 594 LADGGCSGGAYDIII C75 595 FPDLGVRVCEKMALY C76 596 PDLGVRVCEKMALYDC77 597 DLGVRVCEKMALYDV C78 598 KGGKKAARLIVYPDL C79 599 GGKKAARLIVYPDLGC80 600 GKKAARLIVYPDLGV C81 601 KKAARLIVYPDLGVR C82 602 KAARLIVYPDLGVRVC83 603 AARLIVYPDLGVRVC C84 604 ARLIVYPDLGVRVCE C85 605 RLIVYPDLGVRVCEKC86 606 LIVYPDLGVRVCEKM C87 607 IVPDLGVRVCEKMA C88 608 VYPDLGVRVCEKMALC89 609 YPDLGVRVCEKMALY C90 610 AQPGYPWPLYGNEGL C91 611 GQPGYPWPLYGNEGLC92 612 AFCSAMYVGDLCGSV C93 613 AFCSALYVGDLCGSV C94 614 ETVQDCNCSIYPGHVC95 615 EFVQDCNCSIYPGHV C96 616 GVLAGLAYYSMVGNW C97 617 GVLFGLAYFSMVGNWC98 618 DQRPYCWHYAPRPCG C99 619 DQRPYCWYPPRPCG C100 620 TCPTDCFRKHPEATYC101 621 YTKCGSGPWLTPRCL C102 622 LNAACNWTRGERCDL C103 623LNAACNFTRGERCDL C104 624 IAQAEAALENLVVLN C105 625 IAQAEAALEKLVVLH C106626 TRVPYFVRAQGLIRA C107 627 TRVPYFVRAHGLIRA C108 628 HAGLRDLAVAVEPVVC109 629 AAGLRDLAVAVEPIV C110 630 ITWGADTAACGDIIL C111 631ITWGAETAACGDIIL C112 632 GQGWRLLAPITAYSQ C113 633 TAYSQQTRGLLGCII C114634 TAYSQQTRGLLGCIV C115 635 GCIITSLTGRDKNQV C116 636 GCIVVSMTGRDKTQVC117 637 VNGVCWTVYHGAGSK C118 638 KGPITQMYTNVDQDL C119 639KGPITQMYSSAEQDL C120 640 GDSRGSLLSPRPVSY C121 641 GDSRGALLSPRPVSY C122642 SYLKGSSGGPLLCPS C123 643 SYLKGSSGGPVLCPS C124 644 GHAVGIFRAAVCTRGC125 645 GVDPNIRTGVRTITT C126 646 VPHPNIEEVALSNTG C127 647TGEIPFYGKAIPIEV C128 648 TGEIPFYGKAIPLEV C129 649 PTSGDVVVVATDALM C130650 QTVDFSLDPTFTIET C131 651 TLHGPTPLLYRLGAV C132 652 VQNEVTLTHPITKYIC133 653 LYREFDEMEECASHL C134 654 TTLLFNILGGWVAAQ C135 655TTLLNILGGWLAAQ C136 656 PSAASAFVGAGIAGA C137 657 PSAATGFVVSGLAGA C138658 TPCSGSWLRDVWDWI C139 659 VAAEEYVEVTRVGDF C140 660 VAAEEYAEVTRHGDFC141 661 FFTEVDGVRLHRYAP C142 662 FFTELDGVRLHRYAP C143 663FFTWVDGVQIHRYAP C144 664 FFTWLDGVQIHRYAP C145 665 YLVGSQLPCEPEPDV C146666 YLVGSQLPCDPEPDV C147 667 LPTWARPDYNPPLLE C148 668 ASLRQKKVTFDRLQVC149 669 ASLRAKKVTFDRLQV C150 670 HIRSVWKDLLEDTET C151 671IDTTIMAKNEVFCVQ C152 672 VMGSSYGFQYSPGQR C153 673 DCTMLVCGDDLVVIC A235674 ADGGCSGGAYDIIIC A236 675 DGGCSGGAYDIIICD A237 676 GGCSGGAYDIIICDEA238 677 GCSGGAYDIIICDEC A239 678 CSGGAYDIIICDECH A240 679SGGAYDIIICDECHS A241 680 TTILGIGTVLDQAET A242 681 TILGIGTVLDQAETA A243682 ILGIGTVLDQAETAG A244 683 LGIGTVLDQAETAGA A245 684 GIGTVLDQAETAGARA246 685 IGTVLDQAETAGARL A247 686 GTVLDQAETAGARLV A248 687TVLDQAETAGARLVV A249 688 VLDQAETAGARLVVL A250 689 LDQAETACARLVVLA A251690 DQAETAGARLVVLAT A252 691 QAETAGARLVVLATA A253 692 AETAGARLVVLATATA254 693 ETAGARLVVLATATP A255 694 TAGARLVVLATATPP A256 695AGARLVVLATATPPG A257 696 GARLVVLATATPPGS A258 697 ARLVVLATATPPGSV A259698 RLVVLATATPPGSVT A260 699 TSILGIGTVLDQAET A261 700 SILGIGTVLDQAETAA262 701 LGIGTVLDQAETAGV A263 702 GIGTVLDQAETAGVR A264 703IGTVLDQAETAGVRL A265 704 GTVLDQAETAGVRLT A266 705 TVLDQAETAGVRLTV A267706 VLDQAETAGVRLTVL A268 707 LDQAETAGVRLTVLA A269 708 DQAETAGVRLTVLATA270 709 QAETAGVRLTVLATA A271 710 AETAGVRLTVLATAT A272 711ETAGVRLTVLATATP A273 712 TAGVRLTVLATATPP A274 713 AGVRLTVLATATPPG A275714 GVRLTVLATATPPGS A276 715 VRLTVLATATPPGSV B5 716 SGMFDSSVLCECYDA B6717 GMFDSSVLCECYDAG B7 718 MFDSSVLCECYDAGC B8 719 FDSSVLCECDAGCA B9 720DSSVLCECYDAGCAW B10 721 SSVLCECYDAGCAWY B11 722 SVLCECYDAGCAWYE B12 723VLCECYDAGCAWYEL B13 724 LCECYDAGCAWYELT B14 725 CECYDAGCAWYELTP B15 726ECYDAGCAWYELTA B16 727 CYDAGCAWYELTPAE B17 728 YDAGCAWYELTPAET B18 729DAGCAWYELTPAETT B19 730 AGCAWYELTPAETTV B20 731 CCAWYELTPAETTVR B21 732CAWYELTPAETTVRL B22 733 AWYELTPAETTVRLR B23 734 WYELTPAETTVRLRA B24 735YELTPAETTVRLRAY B25 736 DAGCAWYELTPAETS B26 737 AGCAWYELTPAETSV B27 738GCAWYELTPAETSVR B28 739 CAWYELTPAETSVRL B29 740 AWYELTPAETSVRLR B30 741WYELTPAETSVRLRA B31 742 GMFDSVVLCECYDAG B32 743 SGMFDSVVLCECYDA B33 744GMFDSVVLCECYDAG B34 745 MFDSVVLCECYDAGA B35 746 FDSVVLCECYDAGAA B36 747DSVVLCECYDAGAAW B37 748 SVVLCECYDAGAAWY B38 749 VVLCECYDAGAAWYE B39 750VLCECYDAGAAWYEL B40 751 LCECYDAGAAWYELT C154 752 DCTMLVNGDDLWIC C155 753DPTMLVCGDDLVVIC C156 754 DPTMLVNGDDLWIC C157 755 LWARMILMTHFFSIL C158756 LWVRMVLMTHFFSIL C159 757 DLPQIIERLHGLSAF C160 758 DLPQIIQRLHGLSAFC161 759 AVRTKLKLTPIPAAS C162 760 AVRTKLKLTPLPAAS C163 761SGGDIYHSLSRARPR C164 762 SGGDIYHSVSRARPR C165 763 WGENETDVLLLNNTR C166764 GENETDVLLLNNTRP C167 765 WFGCTWMNSTGFTKT C168 766 FGCTWMNSTGFTKTCC169 767 GCTWMNSTGFTKTCG C170 768 GLPVSARRGREILLG C171 769LPVSARRGREILLGP C172 770 PVSARRGREILLGPA C173 771 VSARRGREILLGPAD C174772 GLPVSALRGREILLG C175 773 LPVSALRGREILLGP C176 774 PVSALRGREILLGPAC177 775 VSALRGREILLGPAD C178 776 PDREVLYREFDEMEE C179 777DREVLYREFDEMEEC C180 778 REVLYREFDEMEECA C181 779 EVLYREFDEMEECAS C182780 VLYREFDEMEECASH C183 781 LYREFDEMEECASHL C184 782 YYLTRDPTTPLARAAC185 783 YLTRDPTTPLARAAW C186 784 LTRDPTTPLARAAWE C187 785TRDPTTPLARAAWET C188 786 RDPTTPLARAAWETA C189 787 DPTTPLARAAWETAR C190788 PTTPLARAWETARH C191 789 YYLTRDPTTPLARAA C192 790 YLTRDPTTPLARAAWC193 791 LTRDPTTPLARAAWE C194 792 TRDPTTPLARAAWET C195 793RDPTTPLARAAWETV C196 794 DPTTPLARAAWETVR C197 795 PTTPLARAAWETVRHPeptide ID SEQ ID (Ipep) NO: 1506 796MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVY LLPRRGPRLGVRATRKTSERSQPRGRRQPIPK1526 797 VNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQ WMNRLIAFASRGNHVSPTHYV 1545798 IKGGRHLTFCHSKKKCDELA 1546 799 TVPQDAVSRSQRRGRTGRGR 1547 800YLVAYQATVCARAQAPPPSWD 1551 801 HLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAY 1552 802 YLHAPTGSGKSTKVPVAYAAQGYKVLVLNPSVAATL GFGAY 1553 803GAAVGSICLGKVLVDILAGYGAGVAGAL VAFKIMSGE 1554 804GAAVGSIGLGKVLVDILAGYGAGVAGAL VAFKVMSGE 1555 805GAAVGSIGLGKVLVDILAGYGAGISGAL VAFKIMSGE 1556 806 FTEAMTRYSAPPGDPP 1557807 SSMPPLEGEPGDPDL 1558 808 CGYRRCRASGVLTTS 1559 809 PVNSWLGNIIMYAPT1560 810 PVNSWLGNIIQYAPT 1561 811 SGMFDSSVLCECYDAGCAWYELTPAETTVRLRAY1562 812 SGMFDSSVLCECYDAGCAWYELTPAETSVRLRAY 1563 813SGMFDSVVLCECYDAGAAWYELTPAETTVRLRAY 1564 814SGMFDSVVLCECYDAGAAWYELTPAETSVRLRAY 1565 815FWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAF 1577 816 GEVQVVSTATQSFLAT 1578 817GEVQVLSTVTQSFLGT 1579 818 FTDNSSPPAVPQTFQV 1580 819 FSDNSTPPAVPQTYQV1581 820 NAVAYYRGLDVSVIPT 1587 821 VNLLPGILSPGALVVGVICAAILRRHVGPGEGAVQWMNRLIAFASRGNHVAPTHYV 1588 822 TTILGIGTVLDQAETAGARLVVLATATPPGSVT 1589823 TSILGIGTVLDQAETAGARLVVLATATPPGSVT 1590 824TSILGIGTVLDQAETAGVRLTVLATATPPGSVT 1591 825TTILGIGTVLDQAETAGVRLTVLATATPPGSVT 1592 826FWAKHMWNFISGIQYLAGLSTLPGNPAVASMMAF 1603 827 VFTGLTHIDAHFLSQTKQ 1604 828VVCCSMSYTWTGALITPC 1605 829 VVCCSMSYSWTGALITPC 1606 830VLTSMLTDPSHITAETA 1607 831 VLTSMLTDPSHITAEAA 1613 832ASSSASQLSAPSLRATCTT 1614 833 LTPPHSARSKFGYGAKDVR 1615 834LTPPHSADSKFGYGAKDVR 1616 835 LTPPHSAKSKYGYGAKEVR 1617 836LTPPHSARSKYGYGAKEVR 1618 837 PMGFSYDTRCFDSTVTE 1619 838PMGFAYDTRCFDSTVTE 1620 839 TGDFDSVIDCNTCVTQ 1621 840 TGDFDSVIDCNVAVTQ1622 841 NTPGLPVCQDHLEFWE 1623 842 YLVAYQATVCARAXAPPPSWD 1624 843LEDRDRSELSPLLLSTTEW 1625 844 LEDRDRSQLSPLLHSTTEW 1626 845ASSSASQLSAPSLKATCTT 1627 846 PEYDLELITSCSSNVSVA 1628 847VCGPVYCFTPSPVVVGTTDR 1629 848 GWAGWLLSPRGSRPSWGP 1630 849LLFLLLADARVCACLWM 1631 850 SGHRMAWDMMMNWSPT 1632 851 TGHRMAWDMMMNWSPT1641 852 ITYSTYGKFLADGGCSGGAYDIIICDECHS 1647 853ARALAHGVRVLEDGVNYATGNLPGCSF SIFLLALLSC 1648 854DPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGG 1649 855DPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLGG 1650 856VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL 1651 857VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL 1652 858 XGGRKPARLIVFPDLGVRVCEKMALYDV1653 859 KGGKKAARLIVYPDLGVRVCEKMALYDV 1654 860 IQLINTNGSWHINRTALNCNDSL1655 861 IQLVNTNGSWHINRTALNCNDSL 1656 862 LPALSTGLIHLHQNIVDVQYLYG

For epitope capture, each peptide pool was incubated with solublerecombinant HLA-class II molecules and specific binding was assessed byan SDS-stability assay. The results using the HLA molecules DRB1*0401,DRB1*0404 and DRB1*0101 are shown in FIGS. 2 and 3 respectively: 28peptide pools were found which bind to DRB1*0401 molecules: no. 1, 2, 6,7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20 from “row” pools and no.23, 25, 26, 27, 29, 30, 31, 34, 36, 38, 39 and 40 from “column” pools(FIG. 2). 35 peptide pools out of 40 tested were positive in binding toDRB1*0404 molecules (FIG. 3), while all peptide pools showed bindingactivity to DRB1*0101 molecules. By finding the intersections ofreactive pools in the array, potential individual binders weredetermined and re-checked for binding affinity individually.

All individually confirmed peptides are summarized in Table 2. Bindingto DRB1*0401 is shown in FIG. 4: 54 individual peptides were identifiedas ligands of this HLA-type. Often several overlapping 15mers in a rowbound to HLA allowing identification of their core binding regions.Peptide differing only by one or two amino acids representing variants(see Table 1) usually bound both to soluble HLA class II molecules. Such“duplicates” were considered to represent the same epitope. Thus, 31ligands capable to bind to DRB1*0401 were identified, including 11previously known class II epitopes. From the latter, however, only two(A202-A206 and B60-B68) had been known to be restricted to DR4 (seeTable 2). 20 ligands are candidates for novel epitopes. For DRB1*0404,64 binders designated as 28 potential epitopes were determined, 4 ofthem belong to already known epitopes (FIG. 5, Table 2). For DRB1*0101,83 peptides representing 44 potential epitopes were identified (FIG. 6,Table 2). Of those, 7 had been described previously but with differentHLA restriction.

All individually confirmed peptides binding to at least one of the 3above mentioned HLA types were also tested for affinity to DRB1*0701molecules in a peptide-competition assay (FIG. 7, Table 2). Here, 50ligands were identified. Of those, 7 correspond to already known classII epitopes, but only one was described as DRB1*0701 epitope(A202-A206).

TABLE 2 HCV derived peptides binding to soluble HLA class II molecules.About 400 15- to 23-mer peptides derived from conserved regions of HCVwere analyzed by the Epitope Capture Method using pools of up to 20peptides arrayed in matrix format (see FIG. 1) and four different HLAclass II molecules. Specific binding was confirmed for individualpeptides. Known/new SEQ potential ID Binding to DRB1 epitope, HLA ID NO:Peptide Sequences *0101 *0401 *0404 *0701 coverage A120 863NTNGSWHINRTALNC * nb A122 864 NGSWHINRTALNCND * * * new DRB1*0101, A124865 SWHINRTALNCNDSL * *0404, *0701: 45- 55% B25 866 DAGCAWYELTPAETS*** * *** B26 867 AGCAWYELTPAETSV *** *** *** nb new DRB1*0101, B28 868CAWYELTPAETSVRL *** ** *** * *0404, *0701: 45- B30 869 WYELTPAETSVRLRA** * ** nb 55% B46 870 AGAAWYELTPAETTV *** *** nb new DRB1*0101, B48 871AAWYELTPAETTVRL *** *** ** *0404, *0701: 45- 55% B84 872GSIGLGKVLVDILAG * * * B86 873 IGLGKVLVDILAGYA * ** * ** new DRB1*0101,B88 874 LGKVLVDILAGYGAG * ** * *0404, *0701: 45- B92 875LVDILAGYGAGVAGA * nb 55% C106 876 TRVPYFVRAQGLIRA * * * * new DRB1*0101,*0404, *0701: 45- 55% C113 877 TAYSQQTRGLLGCII *** * new DRB1*0101, C114878 TAYSQQTRGLLGCIV *** * * * *0404, *0701: 45- 55% 1627 879PEYDELELITSCSSNVSVA *** *** ** new DRB1*0101, *0404, *0701: 45- 55% 1628880 VCGPVYCFTPSPVVVGTTDR *** ** * * new DRB1*0101, *0404, *0701: 45- 55%1629 881 GWAGWLLSPRGSPRPSWGP * * ** * new DRB1*0101, *0404, *0701: 45-55% 1604 882 VVCCSMSYTWTGALITPC * ** *** new DRB1*0101, *0404, *0701:45- 55% 1630 883 LLFLLLADARVCACLWM * nb *** new DRB1*0101, *0701: 40-50%C97 884 GVLFGLAYFSMVGNW ** ** nb ** new DRB1*0101, *0701: 40-50% 1547885 YLVAYQATVCARAQAPPPSWD *** NB ** new DRB1*0101, *0701: 40-50% B94 886DILAGYGAGVAGALV * nb nb nb B95 887 ILAGYGAGVAGALVA * nb nb newDRB1*0101, B96 888 LAGYCAGVAGALVAF ** ** nb * *0404, *0701: 40- B97 889AGYGAGVAGALVAFK * nb nb 50% B98 890 GYGAGVACALVAKFI * nb nb nb A272 891ETAGVRLTVLATATT * * nb A274 892 AGVRLTVLATATPPG * nb nb new DRB1*0101,A276 893 VRLTVLATATPPGSV nb * *0404, *0701: 40- 50% B120 894AGISGALVAFKIMSG * nb * *** new DRB1*0101, *0404, *0701: ~45 B122 895VNLLPAILSPGALVV * nb * * new DRB1*0101, *0404, *0701: ~45 C108 896HAGLRDLAVAVEPVV * nb * * new DRB1*0101, *0404, *0701: ~45 C134 897TTLLFNILGGWVAAQ * nb * ** new DRB1*0101, *0404, *0701: ~45 C152 898VMGSSYGFQYSPGQR * nb * * new DRB1*0101, *0404, *0701: ~45 1606 899VLTSMLTDPSHITAETA nb *** ** ** new DRB1*0401, *0404, *0701: ~45 1607 900VLTSMLTDPSHITAEAA nb *** ** * new DRB1*0401, *0404, *0701: ~45 1577 901GEVQVVSTATQSFLAT nb * *** *** new DRB1*0401, *0404, *0701: ~45 1578 902GEVQVLSTVTQSFLGT nb * *** *** new DRB1*0401, *0404, *0701: ~45 B50 903AGAAWYELTPAETSV *** *** *** new DRB1*0101, B52 904 AAWYELTPAETSVRL ****** *** nb *0401, *0404: ~40 1623 905 YLVAYQATVCARAKAPPPSWD * ** newDRB1*0101, *0401, *0404: ~40 C130 906 QTVDFSLDPTFTIET ** *** ** nb newDRB1*0101, *0401, *0404: ~40 1603 907 VFTGLTHIDAHFLSQTKQ *** nb nb * newDRB1*0101, *0701: ~40 C96 908 GVLAGLAYYSMVGNW ** nb nb * new DRB1*0101,*0701: ~40 C191 909 YYLTRDPTTPLARAA nb *** nb new DRB1*0401, *0701: ~40A216 910 SGKSTKVPVAYAAQG nb * nb nb A218 911 KSTKVPVAYAAQGYK nb * nb newDRB1*0101, A220 912 TKVPVAYAAQGYKVL * ** nb *0401 ~35 A222 913VPVAYAAQGYKVLVL * nb nb nb A224 914 VAYAAQGYKVLVLNP * nb nb A242 915TILGIGTVLDQAETA nb nb * new DRB1*0101, A244 916 LGIGTVLDQAETAGA * * nbnb *0401: ~35 C92 917 AFCSAMYVGDLCGSV * ** nb nb new DRB1*0101, C93 918AFCSALYVGDLCGSV * * nb *0401: ~35 A174 919 PALSTGLLHLHQNIV nb * * newDRB1*0404, *0701: ~25-30 B32 920 SGMFDSVVLCECYDA * nb *** B34 921MFDSVVLCECYDAGA * nb *** nb new DRB1*0101, B36 922 DSVVLCECYDAGAAW * nb*** *0404: ~20-25 B38 923 VVLCECYDAGAAWYE nb nb * nb B100 924GAGVAGALVAFKIMS ** nb ** new DRB1*0101, B102 925 GVAGALVAFKIMSGE *** nb*** *0404: ~20-25 C135 926 TTLLLNILGGWLAAQ ** nb * new DRB1*0101, *0404:~20-25 C162 927 AVRTKLKLTPLPAAS nb * * nb new DRB1*0401, *0404: ~20-251618 928 PMGFSYDTRCFDSTVTE nb nb ** new DRB1*0701: ~25 1622 929NTPGLPVCQDHLEFWE nb nb *** new DRB1*0701: ~25 1624 930LEDRDRSELSPLLLSTTEW nb nb * new DRB1*0701: ~25 1546 931TVPQDAVSRSQRRGRTGRGR nb nb nb * new DRB1*0701: ~25 1556 932FTEAMTRYSAPPGDPP nb nb nb * new DRB1*0701: ~25 A114 933 LPGCSFSIFLLALLS** nb nb nb new DRB1*0101: ~20 B58 934 MWNFISGIQYLAGLS * nb nb newDRB1*0101: ~20 B112 935 VDILAGYGAGISGAL * B114 936 ILAGYGAGISGALVA ***new DRB1*0101: ~20 B116 937 AGYGAGISGALVAFK *** nb nb B118 938YGAGISGALVAFKIM *** nb B18 939 DAGCAWYELTPAETT *** nb nb B20 940GCAWYELTPAETTVR *** nb new DRB1*0101: ~20 B22 941 AWYELTPAETTVRLR ** nbnb C112 942 GQGWRLLAPITAYSQ ** nb new DRB1*0101: ~20 C116 943GCIVVSMTGRDKTQV * nb nb new DRB1*0101: ~20 C122 944 SYLKGSSGGPLLCPS * nbnb new DRB1*0101: ~20 C127 945 TGEIPFYGKAIPIEV * nb new DRB1*0101: ~20C144 946 FFTWLDGVQIHRYAP ** nb nb new DRB1*0101: ~20 C159 947DLPQIIERLHGLSAF * nb nb nb new DRB1*0101: ~20 C160 948 DLPQIIQRLHGLSAF *nb C174 949 GLPVSALRGREILLG * nb nb new DRB1*0101: ~20 1558 950CGYRRCRASGVLTTS *** nb nb new DRB1*0101: ~20 1581 951 NAVAYYRGLDVSVIPT** nb nb nb new DRB1*0101: ~20 C95 952 EFVQDCNCSIYPGHV nb ** nb nb newDRB1*0401: ~20 C129 953 PTSGDVVVVATDALM nb nb ** new DRB1*0404: ~5 C157954 LWARMILMTHFESIL nb nb * nb new DRB1*0404: ~5 C158 955LWVRMVLMTHFFSIL nb * A254 956 ETAGARLVVLATATP nb nb * A256 957AGARLVVLATATPPG nb nb * new DRB1*0404: ~5 A258 958 ARLVVLATATPPGSV nb nb** 1605 959 VVCCSMSYSWTGALITPC nb nb * nb new DRB1*0404: ~5 C109 960AAGLRDLAVAVEPIV nb * new DRB1*0404: ~5 C161 961 AVRTKLKLTPIPAAS nb * newDRB1*0404: ~5 A60 962 LGKVIDTLTCGFA ** nb nb ** known DR4, DR8, DR15 A61963 GKVIDTLTCGFAD ** new DR*0101, 0701 A70 964 TCGFADLMGYIPLVG * nb nbA72 965 GFADLMGYIPLVGAP *** * ** known class II A74 966 DLMGYIPVVGAPLGG*** *** ** DR*0101, 0404, 0701 A88 967 CGFADLMGYIPVVGA ** ** * A90 968FADLMGYIPVVGAPL *** *** known class II A92 969 DLMGYIPVVGAPLGG *** ****** DR*0101, 0404, 0701 A96 970 LAHGVRVLEDGVNYA nb *** nb A98 971HGVRVLEDGVNYATG nb *** *** ** known DR11 A100 972 VRVLEDGVNYATGNL nb ****** * new DR*0401, A102 973 VLEDGVNYATGNLPG nb * 0404, 0701 A104 974EDGVNYATGNLPGCS nb * nb nb A200 975 AAQGYKVLVLNPSVA nb *** ** A202 976QGYKVLVLNPSVAAT *** *** *** *** known DRB1*0401, A204 977YKVLVLNPSVAATLG *** *** *** *** 0701, DR11, DR15 A206 978VLVLNPSVAATLGFG *** *** *** *** new DR*0101 C30 979 AVQWMNRLIAFASRG * nbknown DR11, DQ5, also DR*0101 B60 980 NFISGIQYLAGLSTL ** nb 8 B62 981ISGIQYLAGLSTLPG *** *** ** B64 982 GIQYLAGLSTLPGNP *** *** ** knowDR#0401, B66 983 QYLAGLSTLPGNPAI * *** ** 1101 B68 984 LAGLSTLPGNPAIAS **** ** new 0101, 0404, 0701 C124 985 GHAVGIFRAAVCTRG ** * nb ** knownDR*0101, 0401, 0701 1620 986 TGDFDSVIDCNTCVTQ nb nb * new DR*0404 1621987 TGDFDSVIDCNVAVTQ * nb nb known DR13, llso DR*0101, 0401 1631 988SGHRMAWDMMMNWSPT nb nb known class II, also DR*0401 1632 989TGHRMAWDMMMNWSPT nb * known class II, also DR*0401 *** strong binding **intermediate binding * weak binding nb no binding Boldface peptide IDsindicates HLA-ligands with confirmed immunogenicity in HLA-transgenicmice Boldface peptide sequences indicate putative core binding regionsbased on prediction algorithms as described in the test. ¹⁾immunogenicin DRB1 *0401 transgenic mice.

Some of the highly promiscuous peptides and/or with computer algorithm(SYFPEITHI (SEQ ID NO:203), TEPITOPE (SEQ ID NO:204))-predictedaffinities were checked for binding to soluble HLA-DRB1*1101 moleculesin a peptide-competition assay as it is described for HLA-DRB1*0701.Several known DR11 epitopes were used as controls and were confirmed tobind HLA-DRB1*1101 molecules in vitro. Among newly identifiedHLA-DRB1*1101 binders, there are peptides with IDs A120, A122, A141,C114, C134, 1426, 1628, 1629 of high affinity, 5 peptides with IDs C106,C135, 1578, 1547, 1604 of moderate affinity and 4 peptides with IDs B46,B48, B86, B96 of weak affinity ligands.

In summary eight novel ligands binding at least to HLA-DRB1*0101, *0401,*0404, *0701 and *1101 (Tab. 2: peptide Ids A120, A122, A141, 1604,1547, 1628, 1629, and Tab. 6: peptide ID 1426); novel 10 ligands bindingat least to HLA-DRB1*0101, *0401, *0404 and *0701 (Tab. 2: peptide IDsA120-A124, B25-B30, B46-B48, B84-B92, C106, C113-C114, 1627, 1628, 1629,1604); 5 novel ligands binding at least to HLA-DRB1*0101, *0401 and*0701, 5 novel ligands binding at least to HLA-DRB1*0101, *0404 and*0701, 4 novel ligands binding at least to HLA-DRB1*0401, *0404 and*0701, 3 novel ligands binding at least to HLA-DRB1*0101, *0401 and*0404, 2 novel ligands binding at least to HLA-DRB1*0101 and *07.01, 1novel ligand binding at least to HLA-DRB1*0401 and *0701, 3 novelligands binding at least to HLA-DRB1*0101, *0401, 1 novel ligand bindingat least to HLA-DRB1*0404 and *0701, 4 novel ligand binding at least toHLA-DRB1*0101 and *0404, 5 novel ligands binding at least toHLA-DRB1*0701, 13 novel ligands binding at least to HLA-DRB1*0101, 1novel ligand binding at least to HLA-DRB1*0401, and 6 novel ligandsbinding at least to HLA-DRB1*0404.

Moreover, 12 known HLA class II epitopes were confirmed, in severalcases binding to alleles not reported yet was demonstrated (Tab. 2, lastgroup).

Having established physical binding too HLA class II it isstraightforward to verify immunogenicity for a given ligand: forinstance peptide IDs A120-A124, B46-B48, 1627, 1604, 1630, 1547, 1623,B112-118, 1558, all binding to one or more HLA class II alleles werealso shown to be immunogenic in HLA-DRB1*0401 transgenic mice (seeExample II).

To determine the optimal epitope within a longer polypeptide, mice canbe vaccinated with a longer polypeptide incorporating the candidateepitope sequences. Generation of specific CD4+ T cell responses againstnaturally processed and presented epitopes can then be assayed byre-stimulation of murine splenocytes or lymph node cells withoverlapping 15-mers and IFN-gamma ELIspot. Final confirmation/validationof the newly identified HLA-ligands can be achieved by testing thesepeptides with T-cells from humans. Ideally, these comprise therapyresponders or subjects spontaneously recovered from infection.

Example II Immunogenicity of HCV-Derived Peptides in HLA-Transgenic Mice

Synthetic HCV-derived peptides (from conserved regions) wereinvestigated for immunogenicity in HLA-transgenic mice: 36 of 68peptides tested were found to induce peptide-specificIFN-gamma-producing cells in vaccination experiments. As summarized inTable 3, some peptides were either immunogenic (+, less than 100peptide-specific cells among a million splenocytes) or even stronglyimmunogenic (++, more than 100 peptide-specific cells among a millionsplenocytes) in DR4- and/or A*0201-transgenic mice.

TABLE 3 SEQ ID Ipep DRB1*0401 A*0201 B*0702 NO Sequence 1506 + 990MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQ PRGRRQPIPK1526 + + + 991 VNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYV1545 + 992 IKGGRHLIFCHSKKKCDELA 1547 ++ +++ 993 YLVAYQATVCARAQAPPPSWD1552 + + 994 YLHAPTGSGKSTKVPVAYAAQGYKVLVLNPSVAATLGFGAY 1553 + + 995GAAVGSIGLGKVLVDILAGYGAGVAGALVAFKIMSGE 1555 + + + 996GAAVGSIGLGKVLVDILAGYGAGISGALVAFKIMSGE 1558 ++ + ++ 997 CGYRRCRASGVLTTS1559 + ++ 998 PVNSWLGNIIMYAPT 1560 + ++ 999 PVNSWLGNIIQYAPT 1562 + 1000SGMFDSSVLCECYDAGCAWYELTPAETSVRLRAY 1563 + + 1001SGMFDSVVLCECYDAGAAWYELTPAETTVRLRAY 1565 ++ ++ + 1002FWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAF 1577 + + 1003 GEVQVVSTATQSFLAT 1578 +1004 GEVQVLSTVTQSFLGT 1580 + 1005 FSDNSTPPAVPQTYQV 1587 + + 1006VNLLPGILSPGALVVGVICAAILRRHVGPGEGAVQWMNRLIAFASRGNHVAPTHYV 1592 ++ ++ +1007 FWAKHMWNFISGIQYLAGLSTLPGNPAVASMMAF 1604 ++ ++ ++ 1008VVCCSMSYTWTGALITPC 1605 + + + 1009 VVCCSMSYSWTGALITPC 1615 + 1010LTPPHSAKSKFGYGAKDVR 1616 + 1011 LTPPHSAKSKYGYGAKEVR 1617 + 1012LTPPHSARSKYGYGAKEVR 1621 + + + 1013 TGDFDSVIDCNVAVTQ 1623 + + + 1014YLVAYQATVCARAKAPPPSWD 1624 + 1015 LEDRDRSELSPLLLSTTEW 1625 + 1016LEDRDRSQLSPLLHSTTEW 1627 + + ++ 1017 PEYDLELITSCSSNVSVA 1628 ++ 1018VCGPVYCFTPSPVVVGTTDR 1630 ++ ++ 1019 LLFLLLADARVCACLWM 1631 + + 1020SGHRMAWDMMMNWSPT 1632 + 1021 TGHRMAWDMMMNWSPT 1641 + 1022ITYSTYGKFLADGGCSGGAYDIIICDECHS 1647 + + 1023ARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSC 1649 + 1024DPRHRSRNVGKVIDTLTCGFADLMGYIPVVGAPLGG 1650 ++ ++ + 1025VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL 1651 ++ ++ + 1026VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL 1652 ++ + 1027KGGRKPARLIVFPDLGVRVCEKMALYDV 1653 + 1028 KGGKKAARLIVYPDLGVRVCEKMALYDV1654 ++ + 1029 IQLINTNGSWHINRTALNCNDSL 1655 ++ + 1030IQLVNTNGSWHINRTALNCNDSL 1656 + 1031 LPALSTGLIHLHQNIVDYQYLYG

Peptide 1526, 1565, 1631, also shown to be immunogenic in HLA-DRB1*0401transgenic mice contain known class II epitopes. Peptide IDs 1526, 1553,1565, 1587, 1623, 1630 also shown to be immunogenic in HLA-A*0201transgenic mice contain known A2epitopes.

For further characterizing the novel epitopes provided herewith, one maydefine the exact HLA restriction of these epitopes and the minimalepitopes within the sequences recognized by T cells. Both can be done bya variety of well-established approaches known to the one skilled in theart (Current Protocols in Immunology, John Wiley & Sons, Inc.).

First, publicly available programs can be used to predict T cellepitopes on the basis of binding motifs. These include for instance:http://bimas.dcrt.nih.gov/molbio/hla_bind/(Parker et al. 1994),http://134.2.96.221/scripts/MHCServer.dll/home.htm (Rammensee at al.1999), http://mypage.ihost.com/usinet.hamme76/(Sturniolo et al. 1999).The latter prediction algorithm offers the possibility to identifypromiscuous T helper-epitopes, i.e. peptides that bind to several HLAclass II molecules. These predictions can be verified by testing ofbinding of the peptide to the respective HLA.

A way of quickly discerning whether the response towards a peptide isclass I or class II restricted is to repeat the ELIspot assay with pureCD4+ or CD8+ T cell effector populations. This can for instance beachieved by isolation of the respective subset by means of magnetic cellsorting. Pure CD8+ T cells can also be tested in ELIspot assays togetherwith artificial antigen-presenting-cells, expressing only one HLAmolecule of interest. One example are HLA-A*0201 positive T2 cells(174CEM.T2, Nijman et al., 1993). Alternatively, one can use ELIspotassays with whole PBMCs in the presence of monoclonal antibodiesspecifically blocking either the CD4+ or CD8+ T cell sub-population.Exact HLA restriction can be determined in a similar way, using blockingmonoclonal antibodies specific for a certain allele. For example theresponse against an HLA-A24 restricted epitope can be specificallyblocked by addition of an HLA-A24 specific monoclonal antibody.

For definition of the minimal epitopes within the peptide sequencesrecognized by T cells, one can test overlapping and truncated peptides(e.g. 8-, 9-, 10-mers) with splenocytes from immunized transgenic miceor T-cells from humans recognizing the respective epitope.

Example III HLA Restriction of Immunogenic HCV-Derived PeptidesInvestigated in Transgenic Mice

Groups of 5 mice (HLA-A*0201-, HLA-DRB1*0401- and HLA-B*0702 transgenicmice, male, 8-14 weeks of age) were injected subcutaneous into the hindfootpads with 100 μg of peptide +IC31 per mouse (50 μg per footpad).(PCT/EP01/12041, WO 02/32451 A1 and PCT/EP01/06433, WO 01/93905 A1; IC31is a combination of the immunizer disclosed in WO 01/93905 and WO02/32451).

6 days after vaccination single cell suspension of pooled spleens wereprepared and additionally pure fractions of CD8+ in the case of A2 andB7 tg mice (CD8+ fraction for B7 mice containing 97% of CD8 and 1.5% ofCD4 cells and for A2 tg mice 83% of CD8 and 8% of CD4 cells) and CD4+for DR4tg mice (CD4+ fraction for DR4tg mice containing 98% of CD4 cellsand 0.2% of CD8 cells) were separated from the spleen cell suspensionusing MACS separating kit (Miltenyi, Germany). All cells (not separatedcells, positive and corresponding negative fractions) were re-stimulatedex vivo with relevant peptide (for instance Ipep1604) and irrelevantpeptides as negative control (known HLA-DRB1*0401 CMV-derived epitopeIpep 1505, HLA-B*0702 HIV-derived epitope Ipep 1787, or HLA-A*0201tyrosinase-derived epitope Ipep1124) to detect INF-γ producing cells inELISpot assay.

As an example shown in FIG. 8-10 the Ipep1604 (VVCCSMSYTWTGALITPC (SEQID NO:30), in combination with immunizer IC31) was able to induce highnumbers of specific INF-γ producing T cells in all three transgenicclass I and II mouse strains. This was shown not only with whole spleenderived cells but also with enriched fractions of CD8+ cellscorrespondingly for A2 and B7 and CD4+ cells for DR4tg mice. Similar,albeit weaker responses were seen with Ipep1605 (VVCCSMSYSWTGALITPC (SEQID NO:126)), a sequence variant with a serine instead of a threonine.

Thus, Ipep1604 contains class I epitopes for HLA-A*0201 and HLA-B*0702and a class II epitope for HLA-DRB1*0401 molecules.

As shown in Tables 2 and 6, Ipep 1604 binds to class II molecules in apromiscuous manner. Thus, it contains further epitopes, at least forHLA-DRB1*0101, DRB1*0404, DRB1*0701 and DRB1*1101.

Other peptides were analysed in a similar way: Ipeps 1605, 1623, 1547,1558, 1559, 1560, 1565, 1592, 1650, 1654 and 1655 were confirmed tocontain human HLA-DRB1*0401 epitopes. Again, for most of these epitopesbinding is not limited to HLA-DRB1*0401 as shown in Tables 2 and 6.

Ipeps 1565, 1605 and 1650 were confirmed to contain human HLA-A*0201epitopes.

Ipeps 1506, 1587 were confirmed to contain human HLA-B*0702 epitopes.

Ipep 1843 with sequence LPRRGPRL (SEQ ID NO:1046) was shown to be theHLA-B*0702 minimal epitope contained in 1506:

FIG. 10 shows mouse IFN-gamma ELIspot with splenocytes or separated CD8+or CD4+ cells from HLA-A*0702 transgenic mice vaccinated withIpep1506+IC31 or Ipep1835+IC31.

FIG. 10 A) and B) shows that after a single vaccination with either Ipep1506+IC31 or Ipep1835+IC31, upon restimulation with overlapping 15mers,the 15mers A30 to A37 (see Tab.1) react. The common sequence of these15mers is LPRRGPRL (SEQ ID NO:1046) (Ipep 1843, see Tab.4).

FIG. 10 C) confirms these findings: after a single vaccination witheither Ipep1506+IC31 or Ipep14835+IC31, significant interferon-gammainduction against Ipep14843 can be detected. In both cases Ipep 1790 anHIV NEF-derived HLA-B*0702 epitope (sequence RPMTYKAAL (SEQ ID NO:1032))was used as negative control for restimulation.

Ipep 1838 with sequence SPGALVVGVI (SEQ ID NO:1045) (see Tab.4) wasshown to be an HLA-B*0702 minimal epitope contained in 1587: In the caseof Ipep14587 a different approach was taken: the sequence of Ipep14587was inspected for HLA-B*0702 binding motifs and a couple of shortpeptides were synthesized accordingly. These were tested in acompetition-type peptide binding assay using soluble HLA-B*0702 and theFITC-labelled reference peptide LPCVLWPVL (SEQ ID NO:1033), which is aknown HLA-B*0702 epitope derived from EBV (Stuber et al., 1995). PeptideIpep14838 showed ˜30% competition when used in 80-fold molar excess for48 h at 37° C. Thus it is likely to present the minimal HLA-B*0702epitope contained in Ipep 1587.

Example IV Identification and Confirmation of Novel HCV PeptidesReactive in IFN-Gamma ELIspot with Human PBMC from HCV TherapyResponders or Patients with Spontaneous Recovery

40 peptide mixtures in matrix format (FIG. 1) containing syntheticpeptides derived from conserved regions of HCV (Table 1) were screenedin IFN-gamma ELIspot using PBMC from more than 50 individuals who wereeither responders to interferon/ribavirin standard therapy, or, who hadspontaneously cleared HCV (i.e. all subjects were HCV antibody positive,but HCV-RNA negative). PBMC from such individuals are supposed tocontain the relevant T-cell populations responsible for clearing HCV.Thus, peptides discovered or confirmed by using these PBMC are likely torepresent the structural determinants of immune protectionagainst/clearance of HCV. Based on the results from this primarymatrix-screen, a number of peptides were chosen for individualre-testing in IFN-gamma ELIspot using PBMC from selected donors. Inaddition, several new peptides incorporating sequences from overlappingreactive peptides or avoiding critical residues like cystein weresynthesized. These are summarized in Table 4.

TABLE 4 additional peptides derived from conserved regions of HCV.Peptide Peptide sequence SEQ ID ID (1 amino acid code) NO: 1006   MWNFISGIQYLAGLSTLPGN 1034 1334 HMWNFISGI 1035 1425          NFISGIQYLAGLSTLPGNPA 1036 1426 HMWNFISGIQYLAGLSTLPGNPA 10371798 IGLGKVLVDILAGYGAGVAGALVAFK 1038 1799 AAWYELTPAETTVRLR 1039 1800DYPYRLWHYPCTVNYTIFKI 1040 1836 DYPYRLWHYPCTVNFTIFKI 1041 1801  AYSQQTRGLL 1042 1827 TAYSQQTRGLLG 1043 1829 SMSYTWTGALITP 1044 1838SPGALVVGVI 1045 1843 LPRRGPRL 1046Results of the secondary screening with individual peptides aresummarized in Table 5. Altogether ˜20% of subjects (G05, G18, H02, H03,H04, H10, H12, H19, H32, H38) showed a significant IFN-gamma T-cellresponse against one ore more of the peptides. In some cases theobserved number of ELIspots was clearly elevated, but not statisticallysignificant above background. In these cases, PBMC (donors H03, H10,H33, H38) were stimulated with the respective peptides in vitro (2rounds of in vitro priming, see Material & Methods) in order to increasethe peptide specific response. Several peptides were confirmed in thisway, results are again summarized in Table 5.

Peptides A3-A7 represent overlapping 15mers spanning the sequenceTNPKPQRKTKRNTNRRPQD (SEQ ID NO:1047). Since they all react with PBMCfrom donor H03, the minimal sequence of the epitope is located withinthe sequence PQRKTKRNTNR (SEQ ID NO:1048). Prediction algorithmsindicate that QRKTKRNTN (SEQ ID NO:1049) and QRKTKRNT (SEQ ID NO:1050)represent ligands of HLA-B*08, whereas RKTKRNTNR (SEQ ID NO:1051) mostprobably binds to HLA-B*2705.

Peptides C64-C70 represent overlapping 15mers spanning the sequenceKGGRKPARLIVFPDLGVRVCE (SEQ ID NO:1052). C64 and C70 react with PBMC fromdonor H32 and H38, respectively. The minimal sequence of the epitope istherefore located within the sequence ARLIVFPDL (SEQ ID NO:1053).Prediction algorithms indicate that ARLIVFPDL (SEQ ID NO:1053)represents a ligand of HLA- HLA-B*2705 and HLA-B*2709.

TABLE 5 Summary of HCV peptides reactive with PBMC. Numbers representpeptide-specific IFN-gamma secreting T- cells/10⁶ PBMC calculated fromELIspot results (duplicate determinations); values >8 (>3x overbackground) were regarded statistically significant. Donors H32 and H33are spontaneously recovered patients. Donors reactive directly ex vivoin IFN-gamma ELIspot with . . . reactive after 2 rounds HCV-derivedpeptides of in vitro stimulation Peptide H32 H33 ID G05 G18 H02 H03 H04H10 H12 H19 SPR H38 H03 H10 SPR H38 1557 20 75 1577 15 30 165  1579 4535 75 1605 25 1615 30 325  1624 30 55 30 420  1628 40 45 25 20 100  162930 70 15 1798 25 115  1799 20 90 1800 35 95 1801 20 20 A3 80 A4 15 A5110  A7 70 A78 25 A170 35 A212 60 A241 35 B08 55 B38 30 B76 35 C64 30C70 20 C92 25 C94 25 C97 35 C98 70 C100 60 70 C101 50 C102 20 C106 45C112 20 C118 35 C120 25 45 105  C134 20 C138 30 . . . reactive after 2Donors reactive directly ex vivo in IFN-gamma ELIspot rounds of in vitrowith HCV-derived peptides stimulation Peptide H32 H33 ID G05 G18 H02 H03H04 H10 H12 H19 SPR H38 H03 H10 SPR 1557 20 75 1577 15 30 165  1579 4535 75 1605 25 1615 30 325  1624 30 55 30 420  1628 40 45 25 20 1629 3070 15 1798 25 115  1799 20 90 1800 35 95 1801 20 20 A3 80 A4 15 A5 110 A7 70 A78 25 A170 35 A212 60 A241 35 B08 55 B38 30 B76 35 C64 30 C70 20C92 25 C94 25 C97 35 C98 70 C100 60 70 C101 50 C102 20 C106 45 C112 20C118 35 C120 25 45 105 

Example V Binding of HCV Derived Peptides to HLA class II Molecules

In addition to the peptides listed in Table 1, several new peptidesincorporating sequences from overlapping reactive peptides or avoidingcritical residues like cystein were synthesized (Table 4). These wereretested for their affinities to class II soluble HLA molecules, andresults were compared to those obtained with the original (Table 6).

TABLE 6 Binding of selected HCV-derived peptides and their 15- mercounterparts to soluble HLA class II molecules (“+++” strong affinity,“++” intermediate affinity, “+” weak affinity, “−” no affinity, “nd” notdone; core binding motifs are underlined) Peptide ID HLA-DRB1* SEQ IDBinding to soluble NO: Peptide sequences 0101 0401 0404 0701 1101 10541798   IGLGKVLVDILAGYGAGVAGALVAFK − − + ++ +/− 1055 B84GSIGLGKVLVDILAG + + + − 1056 B86   IGLGKVLVDILAGYG + ++ + + +/− 1057 B88    LGKVLVDILAGYGAG + ++ + 1058 B92         LVDILAGYGAGVAGA + − 1059 B94          DILAGYGAGVAGALV + − − − 1060 B96             LAGYGAGVAGALVAF++ ++ − +/− +/− 1061 1799   AAWYELTPAETTVRLR +++ + + − +/− 1062 B46AGAAWYELTPAETTV +++ +++ +++ − +/− 1063 B48   AAWYELTPAETTVRL +++ +++ +++− +/− 1064 1827 TAYSQQTRGLLG ++ − +/− + + 1065 C114 TAYSQQTRGLLGCIV ++++/− +/− + ++ 1066 1829     SMSYTWTGALITP + − − + +/− 1067 1604VVCCSMSYTWTGALITPC + + ++ ++ + 1068 1650VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL 1069 A130  DYPYRLWHYPCTVNF + ++ +/−1070 A131   YPYRLWHYPCTVNFT − 1071 A135       LWH YPCTVNFTI FKV − − ++1072 A141             TVNFTIFKVRMYVGG − − +/− ++ 1073 A145                TIFKVRMYVGGVEHR +/− − 1074 1651VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL 1075 1800  DYPYRLWHYPCTVNYTIFKI − − +/−++ − 1076 A147  DYPYRLWHYPCTVNY − − 1077 A152       LWHYPCTVNYTIFKI − −1078 A158             TVNYTIFKIRMYVGG − − +/− 1079 A162                TIFKIRMYVGGVEHR +/− − 1080 1817                   RMYVGGVEHRL − − +/− 1081 1426 HMWNFISGIQYLAGLSTLPGNPA + + ++ ++ + 1082 1425     NFISGIQYLAGLSTLPGNPA++ ++ ++ nd nd 1083 1006   MWNFISGIQYLAGLSTLPGN ++ + ++ nd nd

Abolished affinities to DRB1*0101 and DRB1*0401 molecules in the case ofpeptide 1798 in comparison with its shorter counterparts (B84-B96) isprobably due to the long sequence (26 amino acids) which can have asecondary structure that prevents binding. It is to be expected that invivo, upon proteolytic cleavage, peptide 1798 will give rise to twoshorter class II epitopes. Removed cystein (C) residues in peptides 1827and 1829 (derivatives of peptides C114 and 1604, respectively) seem tobe crucial for binding to DRB1*0401 molecules but do not essentiallychange affinities to other tested DR subtypes.

Example VI Identification and Characterization of HCV-Epitope hotspots

Here a T-cell epitope hotspot (thereafter referred to as “hotspot”) isdefined as a short peptide sequence at least comprising more than oneT-cell epitope. For example, two or more epitopes may be located shortlyafter each other (shortly being defined as less than 5-10 amino acids),or directly after each other, or partially or even fully over-lapping.Hotspots may contain only class I or class II epitopes, or a combinationof both. Epitopes in hotspots may have different HLA restrictions.

Due to the highly complex and selective pathways of class I and class IIantigen processing, referred to in the introduction, T-cell epitopescannot be easily predicted within the sequence of a polypeptide. Thoughwidely used, computer algorithms for T-cell epitope prediction have ahigh rate of both false-negatives and false-positives.

Thus, as even individual T-cell epitopes are not obvious within thesequence of a polypeptide, the same is even more the case for hotspots.Several radically different experimental approaches are combinedaccording to the present invention for T-cell epitope identification,including epitope capture, HLA-transgenic animals and in vitrostimulation of human mononuclear cells. All three approaches aresystematically applied on overlapping peptides spanning the antigen ofinterest, enabling comprehensive identification of epitopes (refer toCMV Epitope Capture patent). Upon such a comprehensive analysis, notlimited to a particular HLA allele, but rather unravelling all possiblytargeted epitopes within a population, epitope hotspots may becomeapparent. Within an antigen, only few if any sequences showcharacteristics of hotspots. Thus the identification of a hotspot isalways a surprising event.

T-cell epitope hotspots offer important advantages: Hotspots canactivate and can be recognized by different T-cell clones of a subject.Hotspots (when comprising epitopes with different HLA restriction) caninteract with T-cells from different non HLA-matched individuals.

Epitope-based vaccines, so far have aimed at selected prevalentHLA-alleles, for instance HLA-A2 , which is expressed in about half ofCaucasians. Since other alleles are less frequent, epitope-basedvaccines with broad worldwide population coverage will have to comprisemany different epitopes. The number of chemical entities (for instancepeptides) of a vaccine is limited by constraints originating frommanufacturing, formulation and product stability.

Hotspots enable such epitope-based vaccines with broad worldwidepopulation coverage, as they provide a potentially high number ofepitopes by a limited number of peptides.

TABLE 7 T-cell epitope hotspots in conserved regions of HCV. Hotspots(incl. some variations) are shown in bold, epitopes contained within thehotspots in normal font. Peptide number and sequence, as well asHLA-class I and class II coverage are given. Source data refers toExamples and Tables within this specification, or literature references.peptide ID peptide sequence Class I class II source data 1835KFPGGGQIVGGVYLLPRRGPRLGVRATRK (SEQ ID NO: 1084) A2, A3, B7 DR11 ExampleIII, VI 83 KFPGGGQIVGGVYLLPRRGPRL (SEQ ID NO: 1085) A2      B7 DR11Example VI 1051             YLLPRRGPRL (SEQ ID NO: 1086) A2 Bategay 19951843               LPRRGPRL (SEQ ID NO: 1087) B7 Example III                  GPRLGVRAT (SEQ ID NO: 1088) B7 Koziel 1993                    RLGVRATRK (SEQ ID NO: 1089) A3 Chang 1999 84GYKVLVLNPSVAAT (SEQ ID NO: 1090) DR1,4,7,11 Tab.2: A200-A206 AYAAQGYKVL(SEQ ID NO: 1091) A24 prediction 84EX AYAAQGYKVLVLNPSVAAT (SEQ ID NO:1092) A24 DR1,4,7,11 Example VI 87 DLMGYIPAV (SEQ ID NO: 1093) A2 Sarobe1998    GYIPLVGAPL (SEQ ID NO: 1094) A24 prediction 87EX DLMGYIPLVGAPL(SEQ ID NO: 1095) A2,A24 Example VI 89                 CINGVCWTV (SEQ IDNO: 1096) A2 Koziel 1995 1577 GEVQVVSTATQSFLAT (SEQ ID NO: 1097) DR 4, 7Tab.2 89EX GEVQVVSTATQSFLATCINGVCWTV (SEQ ID NO: 1098) A2 DR 4, 7Example VI 1426 HMWNFISGIQYLAGLSTLPGNPA (SEQ ID NO: 1099) A2 DR1,4,7,11Example VII 1006  MWNFISGIQYLAGLSTLPGN (SEQ ID NO: 1100) Example VII1425    NFISGIQYLAGLSTLPGNPA (SEQ ID NO: 1101) Example VII         QYLAGLSTL (SEQ ID NO: 1102) A24 prediction 1334 HMWNFISGI (SEQID NO: 1103) A2 Wentworth 1996 1650 VDYPYRLWHYPCTVNFTIFKVRMYVGGVEHRL(SEQ ID NO: 1104) Cw7,A2,A24, DR1,4,7,11 Tab. 2,3,6 A11 ,A3 Example III1836  DYPYRLWHYPCTVNFTIFKI (SEQ ID NO: 1105) Cw7,A2;A24, DR1,4,7,11 Tab.2,3,6 A11 1846  DYPYRLWHYPCTVNFTIFKV (SEQ ID NO: 1106) Cw7,A2;A24,DR1,4,7,11 Tab. 2,3,6 A11 Example III 1651VDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRL (SEQ ID NO: 1107) Tab. 2,3,6 1800 DYPYRLWHYPCTVNYTIFKI (SEQ ID NO: 1108) Cw7,A24,A11 DR7 Tab. 2,5,6 1754 DYPYRLWHY (SEQ ID NO: 1109) Cw7 Lauer 2002 1815          TVNYTIFKI (SEQID NO: 1110) A11 prediction 1816          TINYTIFK (SEQ ID NO: 1111) A11Koziel 1995          TVNFTIFKV (SEQ ID NO: 1112) A11 prediction        HYPCTVNYTI (SEQ ID NO: 1113) A24 prediction         HYPCTVNFTI(SEQ ID NO: 1114) A24 prediction                      RMYVGGVEHR (SEQ IDNO: 1115) A3 Chang 1999 1799 AAWYELTPAETTVRLR (SEQ ID NO: 1116) B7? B35DR1, 4 Tab. 2,5,6 1818       TPAETTVRL (SEQ ID NO: 1117) B7? B35 Ibe1998 1827EX   GWRLLAPITAYSQQTRGLLGCIV (SEQ ID NO: 1118) A2,A3,A24,DR1,4,7,11 Example VI B8 C114           TAYSQQTRGLLGCIV (SEQ ID NO:1119) A24, B8? DR1,4,7,11 Tab. 2, 6 1827           TAYSQQTRGLLG (SEQ IDNO: 1120) A24, B8 DR1, 7,11 Tab. 6 C112 GQGWRLLAPITAYSQ (SEQ ID NO:1121) A3?,A2?, DR1 Tab.2, 5     RLLAPITAY (SEQ ID NO: 1122) A3prediction C114EX GQGWRLLAPITAYSQQTRGLLGCIV (SEQ ID NO: 1123)A24,A3?,A2?, DR1,4,7,11 Tab. 2, 5, 6 B8? 1827EX GQGWRLLAPITAYSQQTRGLLG(SEQ ID NO: 1124) A24,A3?,A2?, DR1, 7,11 Tab. 2, 5, 6 B8? 1801       AYSQQTRGLL (SEQ ID NO: 1125) A24 Tab. 5 1819        AYSQQTRGL(SEQ ID NO: 1126) A24 Kurokohchi 2001 1798 IGLGKVLVDILAGYGAGVAGALVAFK(SEQ ID NO: 1127) A2,24,3,11 DR1,4,7 Tab. 2,3,5,6 1820         ILAGYGAGV (SEQ ID NO: 1128) A2 Bategay 1995 1821                 VAGALVAFK (SEQ ID NO: 1129) A3,11 Chang 1999            GYGAGVAGAL (SEQ ID NO: 1130) A24 prediction 1604VVCCSMSYTWTGALITPC (SEQ ID NO: 1131) A2,A24,B7 DR1,4,7,11 Tab. 2,3,61829     SMSYTWTGALITP (SEQ ID NO: 1132) A2,A24,B7, DR1,7,11 Tab. 6    SMSYTWTGAL (SEQ ID NO: 1133) A2,B7 prediction       SYTWTGALI (SEQID NO: 1134) A24 prediction 1579 FTDNSSPPAVPQTFQV (SEQ ID NO: 1135)A1,2;B7,51 DR53 = B4*01 Tab. 5 1624 LEDRDRSELSPLLLSTTEW (SEQ ID NO:1136) A1,2,3,26 DR7 Tab. 2,3,5 B8,27,4402,60 1848 LEDRDRSELSPLLLST (SEQID NO: 1137) A1,2,3,26, DR7 Example VI B8,27,4402,60      RSELSPLLL (SEQID NO: 1138) A1 prediction        ELSPLLLST (SEQ ID NO: 1139) A2,A3prediction   DRDRSELSPL (SEQ ID NO: 1140) A26,B27 prediction LEDRDRSEL(SEQ ID NO: 1141) B08,B4402 prediction 1824 LEDRDRSEL (SEQ ID NO:1142)B60 Wong 2001 1547 YLVAYQATVCARAQAPPPSWD (SEQ ID NO: 1143) A2 DR1,4,7,11Tab. 2,3 1822 YLVAYQATV (SEQ ID NO: 1144) A2 Wentworth 1996 A1A7MSTNPKPQRKTKRNTNR (SEQ ID NO: 1145) A11,B08,B27 Tab. 5 A3A7      PQRKTKRNTNR (SEQ ID NO: 1146) B08,527 Tab.5        QRKTKRNTN (SEQID NO: 1147) B08 prediction         RKTKRNTNR (SEQ ID NO: 1148) B2705prediction MSTNPKPQR (SEQ ID NO: 1149) A11 prediction MSTNPKPQK (SEQ IDNO: 1150) A11 Wong 1998 A122EX LINTNGSWHINRTALNCNDSL (SEQ ID NO: 1151)A2,2,3,B8 DR1,4,7,11 Tab. 2,3 A122     NGSWHINRTALNCNDSL (SEQ ID NO:1152) A2 DR1,4,7,11 Tab. 2,3 LINTNGSWHI (SEQ ID NO: 1153) A2,3prediction        RTALNCNDSL (SEQ ID NO: 1154) A2 prediction 1825LINTNGSWHINRTALN (SEQ ID NO: 1155) A2,3,B8 prediction 1826      SWHINRTALN (SEQ ID NO: 1156) B8 prediction A241 TTILGIGTVLDQAET(SEQ ID NO: 1157) A2,A3 DR1, 4 Tab. 2,5 TTILGIGTV (SEQ ID NO: 1158) A2prediction   TILGIGTVL (SEQ ID NO: 1159) A3 prediction B8B38FDSSVLCECYDAGAAWYE (SEQ ID NO: 1160) A1,2,3,26 Tab. 5 B8 FDSSVLCECYDAGCA(SEQ ID NO: 1161) A3,A26 Tab.5     VLCECYDAGA (SEQ ID NO: 1162) A2prediction B38    VVLCECYDAGAAWYE (SEQ ID NO: 1163) A1 Tab. 5 C70EX      ARLIVFPDLGVRVCEKMALY (SEQ ID NO: 1164) A2,A3,B27 Tab. 5 C64-C70      ARLIVFPDL (SEQ ID NO: 1165) B*2705?,*2709? Tab.5 1831       RLIVFPDLGV (SEQ ID NO: 1166) A2 Gruener 2000 1832                 RVCEKMALY (SEQ ID NO: 11670) A3 Wong 1998 C92AFCSAMYVGDLCGSV (SEQ ID NO: 1168) A2,B51 DR1,4 Tab.2,5 C97GVLFGLAYFSMVGNW (SEQ ID NO: 1169) A2,3,26, DR1,4,7 Tab.5 B2705,51 C106TRVPYFVRAQGLIRA (SEQ ID NO: 1170) A3,24, DR1,4,7 Tab.2,5 B7,B8,B2705C134 TTLLFNILGGWVAAQ (SEQ ID NO: 1171) A2 DR1,7,11 Tab.2,5 1823    LLFNILGGWV (SEQ ID NO: 1172) A2 Bategay 1995

Example VII HCV Epitope Hotspot Ipep 1426 Contains at Least HLA-A*201and Several Promiscuous Class II T-Cell Epitopes

The major objective of this experiment was to compare the immunogenicityof the “hotspot” Ipep 1426, which contains at least one HLA-A*0201epitope (Ipep 1334) and 2 promiscuous class II epitopes (Ipeps 1006 and1425), to the individual epitopes. To this end peripheral bloodmononuclear cells (PBMC) from several healthy HLA-typed blood donorswere stimulated in vitro either with 1426 or a mixture of 1334, 1006,1425. Three rounds of stimulation were performed resulting inoligoclonal T cell lines. Then, responses against all four peptides wereassessed by interferon-gamma (IFN-γ) ELIspot analysis.

Peptide 426, induces T cell responses similarly well as individualepitopes comprised within its sequence. In particular CD8 positive Tcells directed against the HLA-A*0201 restricted epitope 1334 weresuccessfully generated.

TABLE 8 peptide induced IFN-γ secretion of oligoclonal T cell lines.Lines were generated from two HLA-typed healthy individuals by 3 roundsof in vitro priming with either peptide 1426 or a mixture of peptides1006 + 1425 + 1334. The reactivity of CD4 and CD8 positive T cells inthese lines was assessed by IFN-γ ELIspot (“+++” very strong, “++”strong, “+” significant, “−” no IFN-gamma secretion). Donor HLA A*0206,A*01, A*0201, A*03, B7, B60; B27, B50; DRB1*1501, −B1*1302 DRB1*0401,−B1*1402 line line raised raised line raised line raised against againstagainst 1006 + against 1006 + Peptide ID 1426 1425 + 1334 1426 1425 +1334 1006 ++ ++ ++ ++ 1425 +++ +++ +++ ++ 1334 + + − − 1006 + 1425 +1334 ++ ++ ++ ++ 1426 +++ +++ +++ ++ 84 (HCV − − − − derived negativecontrol)

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1. A method for isolating Hepatitis C Virus peptides (HPs) or isolatingHCV T cell epitopes which have a binding capacity to a purified,recombinant MHC/HLA molecule or a complex comprising the HCV-peptide orHCV T cell epitope and the purified, recombinant MHC/HLA molecule, themethod comprising: providing a pool comprising at least 10 HCV peptides,the pool containing HCV-peptides which bind to the purified, recombinantMHC/HLA molecule (binding HCV peptides) and HCV-peptides which do notbind to the purified, recombinant MHC/HLA molecule (non-binding HCVpeptides); contacting the purified, recombinant MHC/HLA molecule withthe pool of HCV-peptides whereby a binding HCV-peptide binds to thepurified, recombinant MHC/HLA molecule and a complex comprising theHCV-peptide and the purified, recombinant MHC/HLA molecule is formed;detecting the complex and, optionally, separating the complex fromnon-binding HCV peptides; optionally isolating and characterizing thebinding HCV peptides from the complex; assaying the complex or theoptionally isolated binding HCV peptides in a T cell assay for T cellactivation capacity; and identifying optionally isolated binding HCVpeptides with T cell activation capacity as a T cell epitope or thecomplex.
 2. The method of claim 1, further comprising separating thecomplex from the HCV-peptides which do not bind to the purified,recombinant MHC/HLA molecule.
 3. The method of claim 1, furthercomprising isolating and characterizing the HCV-peptide from thecomplex.
 4. The method of claim 1, further defined as a method forisolating HPs.
 5. The method of claim 1, further comprising isolatingand characterizing the HCV-peptide from the complex before assaying theHCV-peptide.
 6. The method of claim 1, wherein the pool of peptides isfurther defined as a pool of overlapping peptides, a pool of proteinfragments, a pool of modified peptides, a pool obtained fromantigen-presenting cells, a pool comprised of fragments of cells, a poolcomprised of peptide libraries, a pool of (poly)-peptides generated froma recombinant DNA library, a pool of proteins and/or protein fragmentsfrom HCV, or a combination of any of these.
 7. The method of claim 6,wherein the pool is obtained from a total lysate or fraction ofantigen-presenting cells.
 8. The method of claim 7, wherein the pool isobtained from a fraction eluted from surface or MHC/HLA molecules of theantigen-presenting cells.
 9. The method of claim 6, wherein the pool iscomprised of fragments of HCV-containing cells, tumor cells, or tissuecells.
 10. The method of claim 9, wherein the pool is comprised offragments from liver cells.
 11. The method of claim 6, wherein the poolis generated from a recombinant DNA library derived from pathogens ortumor cells.
 12. The method of claim 1, wherein the purified,recombinant MHC/HLA molecules are HLA class I molecules, HLA class IImolecules, non classical MIHC/HLA molecules, MHC/HLA-like molecules or amixture thereof.
 13. The method of claim 1, wherein the characterizingof the HCV-peptides of the complex comprises at least one of massspectroscopy, polypeptide sequencing, a binding assay, identification ofHCV-peptides by determination of their retention factors bychromatography, or a spectroscopic technique.
 14. The method of claim13, wherein the characterizing of the HCV-peptides of the complexcomprises a binding assay, further defined as an SDS-stability assay.15. The method of claim 13, wherein the characterizing of theHCV-peptides of the complex comprises identification of HCV-peptides bydetermination of their retention factors by HPLC.
 16. The method ofclaim 13, wherein the characterizing of the HCV-peptides of the complexcomprises violet (UV) spectroscopy, infra-red (IR) spectroscopy, nuclearmagnetic resonance (NMR) spectroscopy, circular dichroism (CD)spectroscopy, or electron spin resonance (ESR) spectroscopy.
 17. Themethod of claim 1, further comprising performing a cytokine secretionassay.
 18. The method of claim 17, wherein the cytokine secretion assayis an Elispot assay, intracellular cytokine staining, FACS, or an ELISA.19. The method of claim 1, wherein the T cell assay comprises the mixingand incubation of the complex with isolated T cells and subsequentmeasuring cytokine secretion or proliferation of the isolated T cells.20. The method of claim 1, wherein the T cell assay comprises measuringup-regulation of an activation marker.
 21. The method of claim 20,wherein the activation marker is CD69 or CD38.
 22. The method of claim20, wherein the T cell assay comprises measuring down-regulation of asurface marker.
 23. The method of claim 22, wherein the surface markeris CD3, CD8 or TCR.
 24. The method of claim 1, wherein the T cell assaycomprises measuring up-/down-regulation of mRNAs involved in T cellactivation.
 25. The method of claim 24, further defined as comprisingreal-time RT-PCR.
 26. The method of claim 1, wherein the T cell assay isa T cell assay measuring phosphorylation/de-phosphorylation ofcomponents downstream of the T cell receptor, T cell assay measuringintracellular Ca⁺⁺ concentration or activation of Ca⁺⁺-dependentproteins, T cell assay measuring formation of immunological synapses, orT cell assay measuring release of effector molecules.
 27. The method ofclaim 26, wherein the T cell assay measuresphosphorylation/de-phosphorylation of p56, lck, ITAMS of the TCR and thezeta chain, ZAP70, LAT, SLP-76, fyn, or lyn.
 28. The method of claim 26,wherein the T cell assay measures release of perforin, a granzyme, orgranulolysin.
 29. A method for preparing an immunogenic compositioncomprising Hepatitis C Virus peptides (HPs) or isolated HCV T cellepitopes which have a binding capacity to a MHC/HLA molecule or acomplex comprising the HCV-peptide or HCV T cell epitope and the MHC/HLAmolecule, comprising: providing a pool comprising at least 10 HCVpeptides, the pool containing HCV-peptides which bind to the MHC/HLAmolecule (binding HCV peptides) and HCV- peptides which do not bind tothe MHC/HLA molecule (non-binding HCV peptides); contacting the MHC/HLAmolecule with the pool of HCV-peptides whereby a binding HCV-peptidebinds to the MHC/HLA molecule and a complex comprising the HCV-peptideand the MHC/HLA molecule is formed; detecting the complex and,optionally, separating the complex from non-binding HCV peptides;optionally isolating and characterizing the binding HCV peptides fromthe complex; assaying the complex or the optionally isolated binding HCVpeptides in a T cell assay for T cell activation capacity; identifyingoptionally isolated binding HCV peptides with T cell activation capacityas a T cell epitope or the complex; and formulating the peptide orepitope in an immunogenic composition.
 30. A method for inducing animmune response directed against Hepatitis C Virus with an immunogeniccomposition comprising Hepatitis C Virus peptides (HPs) or isolated HCVT cell epitopes which have a binding capacity to a MHC/HLA molecule or acomplex comprising the HCV-peptide or HCV T cell epitope and the MHC/HLAmolecule, comprising: providing a pool comprising at least 10 HCVpeptides, the pool containing HCV-peptides which bind to the MHC/HLAmolecule (binding HCV peptides) and HCV- peptides which do not bind tothe MHC/HLA molecule (non-binding HCV peptides); contacting the MHC/HLAmolecule with the pool of HCV-peptides whereby a binding HCV-peptidebinds to the MHC/HLA molecule and a complex comprising the HCV-peptideand the MHC/HLA molecule is formed; detecting the complex and,optionally, separating the complex from non-binding HCV peptides;optionally isolating and characterizing the binding HCV peptides fromthe complex; assaying the complex or the optionally isolated binding HCVpeptides in a T cell assay for T cell activation capacity; identifyingoptionally isolated binding HCV peptides with T cell activation capacityas a T cell epitope or the complex; formulating the peptide or epitopein an immunogenic composition; and administering the immunogeniccomposition to a subject, wherein an immune response specific forHepatitis C Virus is induced.
 31. The method of claim 30, wherein thesubject is a human.